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Late sexual maturity and prolonged postpartum ovarian acyclicity are responsible for longer non-productive periods, thereby compromising the economic viability of buffalo farming. Age at first calving is a function of growth, while calving interval relies on nutrient partitioning to restore ovarian functions in conjunction with the onset of lactation. Whole blood transcriptome data obtained from heifers calving late (42.2 months) were compared with those calving at a younger age (25.1 months) to determine differentially expressed genes (DEGs) (p < 0.05, FDR < 0.05) for age at first calving. Similarly, DEGs for calving interval between the first two calvings were identified by comparing transcriptomic data obtained from buffaloes with long (417 days) vs . short (363 days) calving intervals. The relevant transcripts were quantified relative to the reference transcriptome, generated by sequencing 16 buffaloes of different physiological status. The quantified transcripts were mapped onto the Bubalus bubalis (Mediterranean water buffalo) reference genome, determining 171 DEGs for age at first calving (P < 1.18 E-72 to 0.00079) and 180 DEGs for calving interval (2.25 E-19 to 0.00054) by DESEQ2 analysis. Differential gene patterns depicted a higher downregulation than upregulation trend for both traits. Significant biological pathway analysis confirmed the role of physiological prerequisites for embryo establishment in the endometrium and for its subsequent development in the uterus. It was evident that pre-embryonic DNA-binding proteins, such as transcription factors (TFs) and histone-modifying factors, are triggered by specific cell signals, facilitating the growth and differentiation of embryonic cells and leading to the formation of the conceptus, first calving in heifers. DEGs obtained for the calving interval were primarily aligned with energy-balancing metabolic changes, with tissue-specific gene expression modified before the formation of the conceptus. In addition, genes governing fat metabolism and immunity were also identified and involved in cellular proliferation and embryogenesis. This study elucidated the genetic regulation and associated pathways governing age at first calving and calving interval in buffaloes. Genomic variants of genes can be considered potential biomarkers for the efficient reproduction and selective breeding for these two traits in buffaloes.