Hygienic behavior represents one of the most prominent disease resistance mechanisms developed by honeybees. This behavior is known to be influenced by the genotype and environmental factors. Therefore, this study aimed to investigate the effect of SNP-8 polymorphism and environmental factors on hygienic behavior in different Apis mellifera breeds. A total of fourteen colonies and 148 bees from seven different bee breeds were used for the Pin-killed Brood Assay (PKB) test and PCR-RFLP analysis, respectively. The PKB assay revealed a wide range of hygienic behavior, spanning from 68% to 100% across the fourteen colonies. The frequencies of the H (High) and L (Low) alleles were 0.0709 and 0.9291, and the frequencies of the HH (High-High), HL (High-Low), and LL (Low-Low) genotypes were 0.027, 0.088, and 0.855 (respectively) and the population was not in the Hardy-Weinberg equilibrium. Additionally, the effects of genotype, breed, birth year of queen, and colony power on hygienic behavior were found to be significant (p < 0.01). Italian hybrid breeds exhibited the highest levels of hygienic behavior, while Anatolian hybrid breeds demonstrated the lowest. It was also found that colonies with young queens, high colony power, and bees with HL and LL genotypes are more hygienic. A noteworthy finding of this study was the detection of heterozygous individuals (HL), marking the first such observation in this study. Consequently, HL and LL genotypes for hygienic behavior in the apiary could be suggested in the selection program. However, more research with more colonies and genes is needed to increase the effectiveness of selection for hygienic behavioral traits in A. mellifera. The widespread of hygienic colonies plays a critical role in preventing the spread of diseases, contributing significantly to the sustainability of beekeeping.