The present study focused on determining the incidence of chromosomal abnormalities in river buffalo embryos produced in vitro to reveal the causes of the low conception rate of transferred embryos. River buffalo oocytes were collected from 2- to 8-mm ovarian follicles of slaughtered river buffaloes in the Philippines and India, matured in vitro, and inseminated with frozen-thawed river buffalo spermatozoa at a final concentration of 1 × 10⁶ sperm/ml. At 36–40 h of in vitro culture, 2- to 8-cell stage embryos were treated with vinblastine sulphate for 6 to 10h, and prepared as chromosomal samples. The rate of chromosomally normal embryos (diploidy 2n = 50) was 52.2% (48/92), while the incidence of chromosomal abnormalities reached 47.8% (44/92) in the analyzed embryos. Polyploidy was the most frequent abnormality (22 embryos; 23.9%), while the incidences of mixoploid (11 embryos, 12%) and haploid embryos (11 embryos, 12%) were the same. The polyploidy and mixoploidy observed in the present study may be the result of polyspermy. The early-stage river buffalo embryos derived from in vitro production displayed a high incidence of chromosomal abnormalities that might affect their subsequent development.