The accurate classification of Neotropical deer is essential for effective conservation strategies; however, many species within the genus Mazama remain taxonomically unresolved due to morphological similarities and historical uncertainties. Mazama tschudii, originally described from the western Andes of Peru, has been debated due to the absence of a designated type specimen and its resemblance to other brocket species. This study integrates morphological, cytogenetic, and molecular data to clarify its taxonomic status, designate a neotype, and highlight its significance for conservation. A recently collected specimen from La Ramada, Lambayeque, Peru, matched the original description of Wagner (1855), exhibiting a smaller size, darker pelage, and distinct cranial features compared to closely related species such as Mazama americana, M. temama, and gray brockets (Subulo gouazoubira, Passalites nemorivagus). Cytogenetic analysis revealed a diploid number of 2n = 42 and a fundamental number (FN) of 68, with extensive chromosomal rearrangements that distinguish it from other Mazama species and suggest reproductive isolation. Mitogenome analysis placed M. tschudii as a sister species to M. temama, yet distinct from M. americana. Despite this close molecular relationship with M. temama, chromosomal divergence and BAC-FISH results demonstrated independent evolutionary trajectories, with ten centric and four tandem fusions differentiating their karyotypes. These results provide robust chromosomal and genomic evidence to validate M. tschudii as a distinct species under the biological species concept. The formal neotype designation from its historical type locality establishes a definitive taxonomic reference, contributing critical insights into the evolutionary complexity of Neotropical deer and reinforcing the importance of integrative taxonomy in shaping conservation priorities.