The Hspa1b gene is one of the first genes expressed after fertilization, with expression observed in the male pronucleus as early as the one-cell stage of embryogenesis. This expression can occur in the absence of stress and is initiated during the minor zygotic genome activation. There is a significant reduction in the number of embryos developing to the blastocyte stage when HSPA1B levels are depleted, which supports the importance of this protein for embryonic viability. However, the mechanism responsible for allowing expression of Hspa1b during the minor zygotic genome activation (ZGA) is unknown. In this report, we investigated the role of HSF1 and HSF2 in bookmarking Hspa1b during late spermatogenesis. Western blot results show that both HSF1 and HSF2 are present in epididymal spermatozoa, and immunofluorescence analysis revealed that some of the HSF1 and HSF2 proteins in these cells overlap the 4',6'-diamidino-2-phenylindole-stained DNA region. Results from chromatin immunoprecipitation assays showed that HSF1, HSF2, and SP1 are bound to the Hspa1b promoter in epididymal spermatozoa. Furthermore, we observed an increase in HSF2 binding to the Hspa1b promoter in late spermatids versus early spermatids, suggesting a likely period during spermatogenesis when transcription factor binding could occur. These results support a model in which the binding of HSF1, HSF2, and SP1 to the promoter of Hspa1b would allow the rapid formation of a transcription-competent state during the minor ZGA, thereby allowing Hspa1b expression.