To investigate the differences between the lineages of the Czech Fleckvieh cattle in Leptin promoter SNP C963T, 695 Czech Fleckvieh cows (650 from production herds and 45 from the Genetic Resources Program (GR)) were examined using PCR-RFLP. The C and T alleles of Leptin promoter were observed with a predominance of C allele in both groups. The most fre­quent genotypes were CC (63%) in production herds and CT (48%) in the GR population. The present study describes, for the first time, the genetic differences in production herds and GR population in Leptin promoter C963T SNP. Variation within the Czech Fleckvieh population was observed and resulted in an advantage to the GR population. Results presented herein empha­sized the importance of the GR program as a reservoir of genetic diversity for indigenous breeds.