Abstract Background Early post-implantation development, especially gastrulation in primates, is accompanied by extensive drastic chromatin reorganization, which remains largely elusive. Results To delineate the global chromatin landscape and understand the molecular dynamics during this period, a single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) was applied to in vitro cultured cynomolgus monkey (Macaca fascicularis, hereafter referred to as monkey) embryos to investigate the chromatin status. First, we delineated the cis-regulatory interactions and identified the regulatory networks and critical transcription factors involved in the epiblast (EPI), hypoblast, and trophectoderm/trophoblast (TE) lineage specification. Second, we observed that the chromatin opening of some genome regions preceded the gene expression during EPI and trophoblast specification. Third, we identified the opposing roles of FGF and BMP signaling in pluripotency regulation during EPI specification. Finally, we revealed the similarity between EPI and TE in gene expression profiles and demonstrated that PATZ1 and NR2F2 were involved in EPI and trophoblast specification during monkey post-implantation development. Conclusions Our findings provide a useful resource and insights into dissecting the transcriptional regulatory machinery during primate post-implantation development.