Prolactin is a hormone which has an important role in the behavior of bird's broodiness. Several studies have shown that prolactin gene could be a candidate gene for broodiness. The variations in the different parts of this gene, have potential usefulness as genetic markers and could help in the genetic improvement of populations. The aim of this research was to investigate the prolactin gene polymorphism using PCR-RFLP method on some masses Khouzestan native chicken. Therefore, random blood sampling from 100 chickens of native chickens in Khuzestan province breeding and Jihad livestock inputs (Bavi city) was performed. Genomic DNA was extracted from these samples, using modified salting out method. Polymerase chain reaction was used to amplification of 439 bp fragments of prolactin gene promoter by specific primers. Allele frequency for C and T, 0.655 and 0.345 and genotype frequencies of CC, CT and TT 0.41, 0.49 and 0.10 were estimated respectively. Using the chi-square (X²) showed that the locus allele frequencies is not in Hardy-Weinberg equilibrium. The results showed that prolactin gene is polymorphic in the studied area and PCR-RFLP method is proper for genotype determination of prolactin gene chicken studies.