Codon usage bias (CUB) in mitochondrial genomes reflects evolutionary forces such as mutation, selection, and genetic drift, yet its dynamics in early-diverging fungal lineages like Conidiobolus (Zoopagomycota) remain unclear. This study systematically analyzed mitochondrial core protein-coding genes (PCGs) from eight Conidiobolus species to elucidate the drivers of CUB and phylogenomic patterns. Nucleotide composition revealed pronounced AT richness (73.32% ± 3.38%) and low GC3 (13.40% ± 5.11%), indicating a preference for A/T-ending codons. Neutrality and ENC-GC3s plots demonstrated that natural selection, rather than mutation pressure, predominantly shaped codon bias, supported by weak GC12-GC3 correlations (slopes: 0.037–0.335) and significant ENC deviations from mutation-driven expectations. PR2-bias analysis further highlighted a strong bias toward A over T and C over G. Correspondence analysis linked major codon usage variations to GC3s, CAI, and FOP indices. Phylogenetic reconstructions based on relative synonymous codon usage (RSCU) and concatenated mitochondrial sequences revealed discordant topologies, particularly in the placement of C. polytocus and C. polyspermus, suggesting divergent evolutionary trajectories. Optimal codon analysis identified species-specific preferences dominated by A/T termini. These findings underscore natural selection as the primary force driving AT-biased mitochondrial CUB in Conidiobolus, while phylogenomic discordance highlights complex evolutionary pressures in this ecologically diverse fungal genus. This study provides foundational insights into mitochondrial genome evolution and codon adaptation mechanisms in early-diverging fungi.
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