DNA barcoding is a reliable tool for the rapid identification of species, uncover hidden diversity, and for biogeographic investigations. Our data set includes 103 ant samples collected from subtropical areas in Enugu State, southeastern, Nigeria, to assess the diversity, population distribution, and phylogenetic relationship of the ant species using a partial region of the mitochondrial COI gene. The most abundant taxa recorded included Camponotus sp., Pachychondyla sp., Pheidole sp., Pheidole rugaticeps, Crematogaster sp., Monomorium subopacum, and Paratrechina longicornis. Sequence analysis of the heatmap grouped the ant samples into four major clusters (A–D), which largely aligned with subfamily designations. Five subfamilies including Myrmicinae, Formicinae, Ponerinae, Dolichoderinae, and Dorylinae were identified with Myrmicinae (39%) having the highest abundance followed by Formicinae (38%), Ponerinae (20%), Dolichoderinae (2%), and Dorylinae (1%). Formicinae exhibited the highest haplotype diversity (Hd = 0.938), while Myrmicinae showed the highest nucleotide diversity (π = 0.203). Genetic distance revealed that the Myrmicinae subfamily is most closely related to the Formicinae (Nei = 0.275; FST = 0.039). In addition, the Dolichoderinae had a closer association with the Formicinae, suggesting shared evolutionary lineages. This finding offers the first phylogenetic framework for ants in southeastern Nigeria based on DNA barcoding, and reveals that the Myrmicinae and Formicinae subfamilies possess broader habitat adaptation than other groups in this region. It also establishes the baseline for future conservation strategies targeting ant communities in West African tropical regions.