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Simple Summary: Livestock production needs to be improved to ensure global food security and meet the growing demand for food. A sheep's gestation length (the period from conception to birth) is usually about 150 days. We have discovered a novel sheep breed in Chitral, Pakistan, whose individuals may gestate for a relatively shorter period, i.e., 100 days, and yet can give birth to normal lambs. To explore the underlying genetic causes for this unique character we used DNA markers. This approach helped us identify some contrasting genetic variations among short-gestating (Kari-S) and long-gestating (Kari-L) Kari ewes. Two of the total 31 targeted DNA sites demonstrated an association with gestation length in Kari sheep. Following an in-depth analysis and further confirmation in other sheep breeds, these DNA sites may be linked to genes controlling gestating length in sheep. The findings from this study may open ways for faster sheep reproduction with reduced cost. Kari sheep inhabiting the Chitral district of Pakistan show variation in gestation length. In this study, we have analyzed the genetic differences between the three subtypes of Kari sheep (based on variation in gestation length) using microsatellite markers. Kari sheep samples were collected from their breeding tract and were characterized for gestation length and genetic diversity using microsatellite markers. A total of 78 Kari ewes were grouped into three categories based on gestation length (GL), i.e., Kari-S (with a shorter GL), Kari-M (with a medium GL), and Kari-L (with a longer GL). DNA from these samples was used to amplify 31 ovine-specific microsatellite loci through PCR. Of the total 78 Kari specimens, 24 were grouped in Kari-S (GL = 100.7 ± 1.8), 26 were from the Kari-M subtype (GL = 123.1 ± 1.0), and 28 were Kari-L (GL = 143.8 ± 1.5). Microsatellite analysis revealed an association of genotypes at two marker sites (MAF214 and ILSTS5) with variation in GL. A total of 158 alleles were detected across the 22 polymorphic loci with an average of 7.18 alleles per locus. Unique alleles were found in all three subtypes. The highest number of unique alleles was observed in Kari-L (15), followed by Kari-S (10) and Kari-M (8). The results indicated that Kari-S is a genetically distinct subtype (with higher genetic differentiation and distance) from Kari-M and Kari-L. The genetic uniqueness of Kari-S is important for further exploration of the genetic basis for shorter gestation length, and exploitation of their unique values. [ABSTRACT FROM AUTHOR]