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Fibroblast growth factor 2 (FGF2) is expressed in the bovine mammary gland and may play a role in the development and reorganization of the mammary gland. It is also expressed by the uterine endometrium throughout the estrous cycle and early pregnancy. The FGF2 was chosen for this study because it regulates the expression of interferon-tau, a key member of the signal transduction pathway involved in milk production. In previous studies, we reported the association of several genes in this pathway with milk production and health traits in dairy cattle. The objective of this study was to examine the association of FGF2 polymorphisms with milk composition, somatic cell score, and productive life in 3 Holstein cattle populations from the United States and Israel. The pooled DNA sequencing approach was used to identify single nucleotide polymorphisms (SNP) in FGF2. Sequencing of a total of 6.4 kb including 3 exons of the gene revealed only one SNP (A/G) in intron 1 at position 11646. This SNP was investigated for association with production traits in 2,773 individuals from 3 Holstein populations: the granddaughter-design Cooperative Dairy DNA Repository and the daughter-design University of Wisconsin populations from the United States and a daughter-design population from Israel. For both the Israeli and the UW populations, FGF2 variants were associated with fat yield and percentage, somatic cell score, and productive life with significant dominance and complete dominance effects. For the Cooperative Dairy DNA Repository population, no significant associations were observed for the examined traits. Given that FGF2 was chosen for this study because of its role in the interferon-tau signal transduction pathway and was found to be associated with production traits, results suggest that the candidate pathway could be an attractive strategy to search for candidate quantitative trait genes.