Global warming is becoming a greater threat for the agricultural sector, while molecular genetics studies still hold new opportunities, to not only detect heat-tolerant animals, but also to allow for increasing the frequencies of desired genotypes in a certain population. In this study, HSP90AB1 gene associated with heat tolerance was investigated in four cattle breeds known as Zavot (ZAV), Sout Anatolian Yellow (SAY), South Anatolian Red (SAR), and Brown Swiss (BS) reared in Türkiye via Allele-Specific Polymerase Chain Reaction (AS-PCR). 4338T>C mutation of the HSP90AB1 gene yielded a total of three genotypes (CC, CT, and TT) across all cattle breeds in which C allele frequency ranged from 0.34 (SAY) to 0.73 (BS), while T allele frequency varied between 0.27 (BS) and 0.66 (SAY). In BS, CC was the genotype with the highest frequency (0.50), whereas the frequency of CC was lower than CT and TT in the Anatolian breed (ZAV, SAR, and SAY). Similarly, the frequency of TT was higher in native Anatolian breeds than BS (0.05). All the populations studied were in Hardy-Weinberg Equilibrium (HWE) in terms of the HSP90AB1 gene. This study confirmed that the HSP90AB1 gene was polymorphic in four cattle breeds reared in Türkiye. This polymorphism has the potential to allow for improving heat tolerance to maintain animal production in the future via suitable selection studies. Therefore, this polymorphism should be conserved in Anatolian cattle breeds, while other genes related to different environmental stressors may be monitored by further studies.