In adult buffaloes, polymorphism of the MT1 receptor gene has shown to influence the reproductive seasonality. The aim of study was to assess whether the polymorphism of the MTNR1A gene may influence puberty in Mediterranean Italian buffalo. The study was conducted using 50 prepubertal buffalo cows that at the age of 15 months were placed into the group where there was the male. Estrus detection was performed by observing estrous-behaviour and pregnancy checking by palpation per rectum and/or ultrasound between days 40 and 60 post-mating. Also of each animal dates of calving was recorded. From each buffalos a blood sample was collected and used for DNA extraction. PCR analysis was performed using 100-150 ng of DNA to amplify the second exon of the MTNRA1 gene. All PCR products were digested with 2U of enzyme HpaI to highlight the polymorphism at position 82 (characterized by a C to a T substitution) of the MTNR1A gene. Frequency of C and T alleles was respectively 0.42 and 0.58 in the analyzed population which resulted in Hardy Weinberg equilibrium. The genotypic frequency was 28% for genotype C/C, 38% for C/T and 34% for T/T. The registration of reproductive data showed that the first heat is around the age of 20 months and the first calving around 32 months. Our data show that the genotype of the MTNR1A does not influence the onset of reproductive activity in prepubertal buffalo cows.