Heat shock proteins (HSPs) are a group of molecular chaperones that are crucial for the proper folding and assembly of proteins and the protection of somatic and germ cells from damage caused by temperature changes. However, the localization and seasonal variation patterns of HSPs in stallions’ testes remain unclear. Thus, we investigated the expression and seasonal variations in the localization of HSP60 and HSP90 in the testes of stallions. Testes from nine Thoroughbred stallions collected following castration during the non-breeding season (NBS) and two different environmental temperature conditions (normal temperature, NT; March–May and hot temperature, HT; June–August) during the breeding season (BS) were used. Testis tissues were subjected to Reverse transcription-quantitative polymerase chain reaction, Western Blot (WB) analysis and immunofluorescence (IF) assay. The results revealed that the relative abundance of HSP60 and HSP90 mRNA transcripts was significantly upregulated under NT and HT conditions during BS compared with that during NBS conditions. The WB analysis showed that the relative intensity of protein bands was significantly higher in NT and HT conditions during the BS than that of those during the NBS. Immunofluorescence assay showed localization of HSP60 in the cytoplasm of the Leydig cells during both seasons. HSP90 primarily showed immunostaining in undifferentiated spermatogonia and primary spermatocytes in NT and HT conditions and was additionally expressed in the spermatids during the NBS. The spermatogenesis was not monitored in the present study; however, findings of this study suggest that the localization of HSP60 and HSP90 varies in different seasons depending on the temperature, providing insight into the potential mechanisms influencing spermatogenesis during breeding and non-breeding seasons in stallions.