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Simple Summary: Subclinical mastitis represents a significant disease in dairy animals and negatively impacts udder health. Genetics, pathogens, and poor health and management procedures in dairy animals contribute primarily to this disease. Animal genetics plays a major role in disease resistance and susceptibility. Studying single-nucleotide polymorphisms in genes helps to detect resistant and susceptible animals. Transferrin is one of the important glycoproteins involved in the innate immune system that prevents iron availability to pathogens. Several studies have reported polymorphism in the transferrin gene in dairy animals with mastitis, but no detailed information on in silico tools is available for identification and verification. So, this study was carried out to detect polymorphism in the transferrin gene in healthy and mastitic cows, and to use various in silico based tools and software to find the impact of single-nucleotide polymorphisms (SNPs) on animal health. The results indicated three SNPs were located in the transferrin gene of Holstein Friesian cross-bred dairy cows. Moreover, the use of online computational tools provided significant knowledge regarding whether the SNPs would be deleterious, damaging, benign, neutral, or affect protein stability and function. Dairy cattle with a high milk yield are susceptible to many infectious diseases, such as mastitis. Subclinical mastitis (SCM) is the most prevalent form of mastitis that predominantly affects animal health, and causes adverse effects on the quality and quantity of milk. In dairy animals, subclinical mastitis often remains undetected, as no gross changes in udder characteristics are visible. In the present study, 135 Holstein Friesian dairy animals were selected and screened as healthy (n = 25) and mastitic (n = 110) based on diagnostic tests such as the California mastitis test, pH, electrical conductivity, and somatic cell count. In this study, the somatic cell count was used as a gold-standard test in differentiating subclinical mastitis animals from healthy ones. The present study was carried out to study polymorphisms in the bovine transferrin gene in cows (with subclinical mastitis and healthy). For the early detection of resistant/or susceptible animals, a useful marker could be provided by the detection of single-nucleotide polymorphisms (SNPs) in the transferrin gene, which are often associated with mammary innate immune response. The sequencing results revealed three nucleotide substitutions: two transversions (230 A > C, 231 C > A) and one transition (294 A > G) in susceptible cows as compared to disease-free subjects. The nucleotide variations at position 230 (GAC > GCA) and 231 (GAC > GCA) were nonsynonymous, and corresponded to an amino acid change from aspartic acid to alanine; whereas at position 294 (GAA > GAG), the mutation was synonymous. In the present study, many in silico tools were taken into consideration to determine the effect of SNPs on protein structure and function. The PROVEAN tool found the amino acid substitution to be neutral and deleterious. PolyPhen-2 revealed the amino acid variations at positions 320 and 321 to most likely be damaging; and at the 341 position, the variations were benign. The I-Mutant and MUpro tools found that the protein stability decreased for nonsynonymous variations. The SIFT tool revealed the protein function was likely to be affected in nonsynonymous variations, with no change in the case of synonymous ones. Phylogenetic analysis of the bovine transferrin gene revealed a close relation of the CA allele with the Bos taurus transferrin, while the G allele was closely related to a cross of Bos indicus × Bos taurus serotransferrins, followed by the Bison bison transferrin. The least relation was shown by both alleles to Capra hircus, Ovis aries, and Bubalus bubalis. [ABSTRACT FROM AUTHOR] more...