Puberty in cattle is regulated by an endocrine axis, which includes a complex milieu of neuropeptides in the hypothalamus and pituitary gland. The neuropeptidome of hypothalamic-pituitary gland tissue of pre- (PRE) and postpubertal (POST) Bos indicus-influenced heifers was characterized, followed by quantitative analysis of 51 fertility-related neuropeptides in these tissues. Comparison of peptide abundances with gene expression levels allowed assessment of post-transcriptional peptide processing. On the basis of classical cleavage, 124 mature neuropeptides from 35 precursor proteins were detected in hypothalamus and pituitary gland tissues of three PRE and three POST Brangus heifers. An additional 19 peptides (cerebellins, PEN peptides) previously reported as neuropeptides that did not follow classical cleavage were also identified. In the pre-pubertal hypothalamus, a greater diversity of neuropeptides (25.8%) was identified relative to post-pubertal heifers, while in the pituitary gland, 38.6% more neuropeptides were detected in the post-pubertal heifers. Neuro-tissues of PRE and POST heifers revealed abundance differences ( p < 0.05) in peptides from protein precursors involved in packaging and processing (e.g., the granin family and ProSAAS) or neuron stimulation (PENK, CART, POMC, cerebellins). On their own, the transcriptome data of the precursor genes could not predict the neuropeptide profile in the exact same tissues in several cases. This provides further evidence of the importance of differential processing of the neuropeptide precursors in the pituitary before and after puberty.