The aim of this study was to ascertain the RsaI polymorphism of the Prolactin (PRL) gene of 186 organically reared Holstein cattle by PCR-RFLP method, in order to reveal the genetic variation of the population, and to reveal the relationship between PRL/RsaI genotypes and some milk yield traits. In the study, the RsaI^-/- genotype frequency, RsaI^+/+ genotype frequency and RsaI^-/+ genotype frequency of Holstein cattle were determined as 0.26, 0.22 and 0.52, respectively. The RsaI^- gene frequency was 0.52, and the RsaI⁺ gene frequency was 0.48. Hardy-Weinberg genetic equilibrium test showed that the distribution of genotype frequencies was determined to be in genetic equilibrium. The relationships between PRL/RsaI polymorphism and the examined milk yield traits were not significant. As a result, an economic and safe method PCR-RFLP for the detection of genetic markers, was successfully used in the determination of the 3 genotypes (RsaI^-/-, RsaI^-/+, and RsaI^+/+) of Prolactin gene in Holstein cattle reared under organic condition, and the results was similar with previously reported literature.