Guinea pigs are valuable models for human disease research, yet the lack of established pluripotent stem cell lines has limited their utility. In this study, we isolate and characterize guinea pig epiblast stem cells (gpEpiSCs) from post-implantation embryos. These cells differentiate into the three germ layers, maintain normal karyotypes, and rely on FGF2 and ACTIVIN A signaling for self-renewal and pluripotency. Wingless/Integrated (WNT) signaling inhibition is also essential for their maintenance. GpEpiSCs express key pluripotency markers (OCT4, SOX2, NANOG) and share transcriptional similarities with human and mouse primed stem cells. While many genes are conserved between guinea pig and human primed stem cells, transcriptional analysis also reveals species-specific differences in pluripotency-related pathways. Epigenetic analysis highlights bivalent gene regulation, underscoring their developmental potential. This work demonstrates both the evolutionary conservation and divergence of primed pluripotent stem cells, providing a new tool for biomedical research and enhancing guinea pigs' utility in studying human diseases.