Poor estrus expression and anestrus negatively impact the reproductive efficiency of buffaloes. This study aimed to evaluate whether adding equine chorionic gonadotropin (eCG) to an estrous synchronization protocol, followed by timed artificial insemination (AI), could enhance ovulation and pregnancy rates in anestrous buffalo cows under tropical conditions. The study involved 50 lactating Murrah buffalo cows, divided into two groups and was treated with two different estrus synchronization programs for timed insemination. The findings suggest that adding eCG to a progesterone-based estrous synchronization protocol significantly improves the ovulation rates in non-cyclic buffaloes. When combined with timed AI, the pregnancy rates in anestrous buffaloes, whether cyclic or non-cyclic, can be comparable to those achieved in cows inseminated after natural estrus.