The present study was planned to determine the macro-mineral status (calcium, phosphorus and magnesium) in riverine buffaloes suffering with prepartum vaginal prolapse (n= 100) in comparison with control (n= 100) buffaloes kept in irrigated and rain fed (Barani) agro-ecological zones of Punjab, Pakistan. Serum macro-mineral status was determined by spectrophotometer using commercially available kits. Mean serum calcium and phosphorus levels were lower in buffaloes suffering with prepartum vaginal prolapse compared with their healthy counterparts (P< 0.01), while reverse was true for serum magnesium concentration. Mean serum calcium was significantly higher (P< 0.01) and phosphorus was nonsignificantly lower in control and vaginal prolapse affected buffaloes kept in irrigated zone compared to their counterparts in rain fed zone. However, magnesium concentration was non-significantly higher in control buffaloes and significantly lower in vaginal prolapse affected buffaloes belonging to irrigated zone compared to their counterparts in rain fed zone. It may be inferred that deficiency of calcium and phosphorus and higher levels of magnesium may possibly be the contributing factors in causing prepartum vaginal prolapse in buffaloes. The levels of calcium, phosphorus and magnesium were, at least partially, zone dependent.