The aim of this study was to compare serum hormone profiles (progesterone and estradiol-17β) in buffaloes with dystocia and unassisted calving in three agro-ecological zones of Punjab, Pakistan. One hundred and seventy three buffaloes (n=173) with assisted (dystocia) and unassisted calving (normal birth) were sampled for study. The results showed that the buffaloes suffering with dystocia had significantly higher (P<0.05) mean serum progesterone level compared with those having normal calving. The comparison amongst the agro-ecological zones revealed that serum progesterone level of dystocia cases in buffaloes of northern irrigated zone was significantly lower (P<0.05) compared with those in the southern irrigated zone and the arid zone, whereas the latter two did not differ between each other. No difference was observed in serum progesterone levels in normal buffaloes when compared amongst three agro-ecological zones. The serum estradiol-17β profile showed a significant (P<0.05) lower level in buffaloes with dystocia as compared to those with normal calving. Mean serum estradiol-17β level in the buffaloes affected with dystocia in the northern irrigated zone was significantly lower (P<0.05) compared to those in the southern irrigated zone and the arid zone wherein the latter two did not differ between each other.