Sustainability in dairy cattle farms depends on the efficiency of milk yield and reproductive traits. Thus, this study aimed to investigate the effect of the FGF-2/Csp6I gene and major environmental factors on these traits in Holstein-Friesian cattle. A total of 212 whole blood samples were collected from the Vena coccygea of cattle and the data obtained from these samples were used in all statistical analyses. Then, the restriction fragment length polymorphism (RFLP) method (determination of genotypes) was conducted and programs including PopGene (allele and genotype frequencies), Minitab (association analyses) and MTDFREML (variance components and genetic parameters) were used. Alleles A (0.4269) and G (0.5731) as well as genotypes AA (0.174), AG (0.505) and GG (0.321) were found, indicating that the population is polymorphic and in Hardy-Weinberg equilibrium (P > 0.05). The effect of the Csp6I polymorphism of FGF-2 gene on peak milk yield (PMY) (P < 0.01); lactation milk yield (LMY), milking time (MT), 305-day and 200-day lactation milk yield (LMY305 and LMY200), average daily milk yield (ADMY) (P < 0.05); 100-day lactation milk yield (LMY100), age of using in first breeding (AUFB) and number of inseminations per conception (NIPC) (P < 0.10) were significant. The heritability of milk yield traits and the correlation between direct and maternal heritability for reproductive traits were high. Furthermore, the breeding value of PMY was higher for the AA genotype (0.745 ± 0.292) than for the AG genotype (−0.268 ± 0.171) (P < 0.05). As a result, the A allele and AA genotype for the FGF-2/Csp6I gene had an increasing effect on milk yield without compromising reproductive performance in Holstein-Friesian dairy cattle.