Within Enugu and Anambra States, Nigeria, identification of fishes has been based on morphological traits and do not account for existing biodiversity. For DNA barcoding, assessment of biodiversity, conservation and fishery management, 44 fish sampled from Enugu and Anambra States were isolated, amplified and sequenced with mitochondrial cytochrome oxidase subunit I (COI). Twenty groups clustering at 100% bootstrap value including monophyletic ones were identified. The phylogenetic diversity (PD) ranged from 0.0397 (Synodontis obesus) to 0.2147 (Parachanna obscura). The highest percentage of genetic distance based on Kimura 2-parameter was 37.00 ± 0.0400. Intergeneric distances ranged from 15.8000 to 37.0000%. Congeneric distances were 6.9000 ± 0.0140–28.1000 ± 0.0380, with Synodontis as the existing synonymous genus. Confamilial distances in percentage were 16.0000 ± 0.0140 and 25.7000 ± 0.0300. Forty-two haplotypes and haplotype diversity of 0.9990 ± 0.0003 were detected. Nucleotide diversity was 0.7372, while Fu and Li’s D* test statistic was 2.1743 (P < 0.02). Tajima’s D was 0.2424 (P > 0.10) and nucleotide frequencies were C (17.70%), T (29.40%), A (24.82%), G (18.04%) and A + T (54.22%). Transitional mutations were more than transversions. Twenty species (99–100%) were identified with the e-value, maximum coverage and bit-score of 1e−43, 99–100 and 185–1194, respectively. Seventeen genera and 12 families were found and Clariidae (n = 14) was the most dominant among other families. The fish species resolution, diversity assessment and phylogenetic relationships were successfully obtained with the COI marker. Clariidae had the highest number of genera and families. Phylogenetic diversity analysis identified Parachanna obscura as the most evolutionarily divergent one. This study will contribute to fishery management, and conservation of freshwater fishes in Enugu and Anambra States, Nigeria.