The aim of this study was to evaluate the efficacy of the Ovsynch protocol used for fixed-time artificial insemination in buffalo when replacing the last injection of GnRH with hCG in order to increase plasma progesterone (P4) concentrations and therefore increase conception rates. Two-hundred twelve buffaloes were randomly assigned into 2 treatment groups: G-GnRH (Day 0, GnRH; Day 7, PGF2α; Day 9, GnRH; n = 94) and G-hCG (Day 0, GnRH; Day 7, PGF2α; Day 9, hCG; n = 118). All buffaloes were inseminated on Day 10, 16 h after the last hormonal injection. On Day 22, 80 buffaloes were randomly selected for blood collections in order to measure plasma P4 concentration. Pregnancy diagnosis was performed on Day 40 by ultrasonography. Conception rates of G-GnRH and G-hCG animals were 46.8% (44/94) and 50.8% (60/118; P > 0.05), respectively. Plasma P4 concentration was lower in G-GnRH (2.94 ± 1.51 ng/ml) than in G-hCG (4.02 ± 2.34 ng/ml; P < 0.05) animals. The plasma P4 of pregnant buffaloes was higher in G-hCG than in G-GnRH (4.66 ± 1.73 ng/ml vs. 3.54 ± 1.05 ng/ml; P < 0.05). This effect was not observed in non-pregnant animals (G-GnRH = 3.18 ± 2.78 ng/ml vs. G-hCG = 2.19 ± 1.69 ng/ml, respectively; P > 0.05). Pregnant G-GnRH and G-hCG buffaloes had higher plasma P4 concentrations than the non-pregnant buffalo of both groups (3.54 ± 1.05 vs. 2.19 ± 1.69 ng/ml and 4.66 ± 1.73 vs. 3.18 ± 2.78 ng/ml, respectively; P < 0.05). In summary, when using the Ovsynch protocol for FTAI in buffalo, the replacement of the last GnRH administration with hCG increased the plasma P4 concentration. However, no positive effect on the conception rate was observed.