Hygienic behavior (HB) is a crucial biological mechanism in honeybees that is associated with disease resistance. This study aimed to investigate the effect of the SNP18 sequence and environmental factors on the HB of honey bees, using a total of 14 colonies and 148 bee samples from seven different bee breeds. Association analysis revealed that colonies with Italian hybrids (IH) or young queens statistically (p < 0.01) exhibited high hygienic behavior (HHB). HB increased by 71.6% when the number of frames in the colony, representing colony power (CP), increased by one (p < 0.05). In restriction fragment length polymorphism (RFLP) analysis, novel mutations in the MlyI polymorphism of the SNP18 sequence were firstly found in Apis mellifera. In addition, the restriction fragments of the novel variants of the SNP18 HHB and SNP18 low hygienic behavior (LHB) lines were determined by sequencing. In this study, several important findings emerged: Due to one-base differences in the restriction fragment, this sequence could not be genotyped by RFLP. Honeybees could be homozygous (HHB or LHB) or heterozygous (HHB and LHB) for the SNP18. SNP18 sequence could be located in different regions of the chromosome and could only be determined by genome sequencing. Finally, since genotypes cannot be clearly determined, no specific allele or genotype can be recommended for HB selection in beekeeping. Therefore, additional research is required to assess discovered novel variants for genetic selection of HHB for ecological beekeeping, healthy products and sustainability.