This study aimed to determine the differences in chemical composition of pilose antler samples from different geographical origins and species. Three types of pilose antler were selected: red deer antler from Xinjiang (XC), red deer antler New Zealand (ZC), and sika deer antler from Jilin (JC). Various instrumental techniques were employed to determine the basic nutritional components, chemical elements, amino acids, volatile organic compounds, and metabolite profile of pilose antler. Multivariate statistical analysis was further used to identify the chemical markers for distinguishing between the three types of pilose antler. The results showed significant differences in total ash and total carbohydrate contents among these types. XC was relatively rich in chemical elements and presented a unique creamy and nutty flavor. In terms of amino acid scores, JC was superior to the other two types, with valine (8.1%), leucine (8.2%), and lysine (9.2%) accounting for higher proportions of total amino acids than the WHO/FAO recommended values. Lipids, organic acids, and their derivatives were the predominant metabolites in the pilose antler samples. Significant differences in metabolite composition were observed among them, with 355 and 199 discriminative metabolites identified in the positive and negative ion modes, respectively. These findings confirm the influence of geographical origin and species on the chemical composition of pilose antler, providing data support for the evaluation of its quality characteristics and processing suitability.