The present study was undertaken to analyze the relative abundance (RA) of pluripotency-associated genes (NANOG, OCT4, SOX2, c-MYC, and FOXD3) in different grades of immature oocytes and various stages of in vitro-produced buffalo embryos using RT-qPCR. Results showed that the RA of NANOG, OCT4, and FOXD3 transcripts was significantly higher (P < 0.05) in A grade oocytes compared with the other grades of oocytes. The RA of the c-MYC transcript was significantly higher (P < 0.05) in A grade compared with the C and D grades of oocytes, but the values did not differ significantly from the B grade of oocytes. The RA of the SOX2 transcript was almost similar in all grades of the oocytes. The expression levels of NANOG (P > 0.05), OCT4 (P > 0.05), c-MYC (P > 0.05) and SOX2 (P < 0.05) were higher in the blastocysts compared with the other stages of the embryos. Markedly, FOXD3 expression was significantly higher (P < 0.05) in 8–16-cell embryos compared with the 2-cell and 4-cell embryos and blastocyst, but did not differ significantly from the morula stage of the embryos. In the study, the majority of pluripotency-associated genes showed higher expression in A grade immature oocytes. Therefore, it is concluded that the A grade oocytes appeared to be more developmental competent and are suitable candidates for nuclear cloning research in buffalo. In buffalo, NANOG, OCT4, SOX2, and c-MYC are highly expressed in blastocysts compared with the other stages of embryos.