Abstract The p38MAPKs (mitogen-activated protein kinases) signaling contributes a pivotal role in mammalian ovarian follicular development; however, the knowledge regarding their expression in geese remains unresolved. The objective of the current study was to determine the spatio-temporal expression of heat shock protein 27 (HSP27) and mitogen- and stress-activated protein kinase 1 (MSK1) genes in the prehierarchical follicles during geese ovarian development. The prehierarchical follicles samples were harvested from 35- to 37-week-old healthy laying geese. HSP27 and MSK1 relative expression in various sized prehierachical follicles was detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. Follicular wall localization of HSP27 and MSK1 was examined by using immunohistochemistry. Our results at mRNA level indicated that HSP27 was highly expressed in middle white follicles whereas MSK1 was predominantly expressed in small white follicles. The western blotting results for HSP27 and MSK1 were inconsistent with the RT-qPCR results in various stages of prehierachical follicular development but noticeably, HSP27 proteins were still expressed more in middle white follicles while MSK1 proteins were more abundant in primary follicles. At different stages of prehierarchical development, immunodetections in the granulosa and theca cells revealed that HSP27 was intensively localized in middle white follicles while strong detections of MSK1 were observed in large white follicles. These results indicate HSP27 and MSK1 might be associated to the key regulators of folliculogenesis in geese.