Unequal usage of synonymous codons in the gene transcript for an amino acid is known as codon usage bias (CUB). It is a unique property of gene as well as genome. Mutation and natural selection are known to be the major factors that influence CUB. Other factors encompass gene expression level, GC content, codon position, recombination rate, RNA stability and gene length. CUB analysis helps in in-depth understanding of the molecular biology, genetics and genome evolution in a species. We used bioinformatic methods to explore the pattern of CUB in MT-ATP6 gene in different classes of platyhelminthes. The analysis is based on genetic code of translational table 14 of National Center of Biotechnology Information (NCBI) where the codon AAA codes for asparagine and TAA for tyrosine amino acid. The synonymous codon usage order (SCUO), an index of CUB, values in different classes namely cestoda, monogenea, rabditophora, trematoda and turbellaria of platyhelminths were found to be 0.43, 0.32, 0.49, 0.40 and 0.36, respectively which suggest that the codon usage bias of ATP6 gene was low (SCUO < 0.50). Highly significant correlation (p < 0.001) was found between SCUO and various GC contents indicating that GC composition had an influence on CUB. From the relative synonymous codon usage (RSCU) analysis on codons, we found most of the over-represented codons in all the classes were A/T ending types, which suggested that the preferred codons were influenced by compositional constraints. The PR2 plot revealed asymmetric usage of AT and GC bases among the four fold degenerate codon families with greater usage of G and T over A and C. Highly significant correlation (p < 0.001) was found between overall nucleotide composition and its 3rd codon position suggesting that both natural selection and mutation pressure might have influenced the CUB among different classes. Neutrality analysis revealed that natural selection might play a major role. Mutational responsive index (MRI) and translational selection (P2) values elucidated that selection for translational efficiency moderately affected the codon usage bias in MT-ATP6 gene.