BACKGROUND: Investigation of chicken major histocompatibility complex (MHC) genes along with their diversity across native regions and populations, as a genetic resource, can be used in the breeding programs. Important characteristics of MHC genes, such as the association with immunological and production traits, make them exceptional candidates for marker assisted selection. AIMS: The aim of this study was to characterize MHC based on the LEI0258 microsatellite marker to evaluate the genetic variability and diversity within and between Iranian chicken populations. METHODS: Blood samples were collected from six indigenous ecotypes (n=633) and Ross 308 (n=216) as a commercial breed. The MHC variability was determined based on a microsatellite marker located within MHC, LEI0258. Polymerase chain reaction and fragment analysis was used for microsatellite polymorphism detection. RESULTS: Based on the fragment analysis, 7 alleles were found in Ross 308 and 25 alleles across all 6 populations. The population with the maximum genetic diversity was Mazandaran (0.939), while the population with the minimum genetic diversity was Ross 308 (0.794). Out of the 6 Iranian chicken ecotypes, all except Arian and Khorasan, were in Hardy-Weinberg equilibrium (P<
0.05). The genetic variations within (84.92%) and between (15.08%) populations were statistically significant (P<
0.002). CONCLUSION: A significant genetic structure that is not completely homogeneous among the Iranian chicken populations can be considered as distinct genetic resources. This study highlights the value of using markers such as LEI0258 to investigate the diversity of genes that play dual roles in immunity and production.