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Ipomoea plants possess important commercial, medicinal, and ornamental value. Molecular and morphological studies have confirmed that most species of this genus exhibit similar phenotypes but complex phylogenetic relationships. To date, limited information is available on these evolutionary relationships. In this study, systematic analysis of diverse species from Ipomoea was used to elucidate the relationships in this genus. To this end, we employed the concept of codon usage bias (CUB) to analyze the codon usage bias of five Ipomoea species such as effective number of codons (ENC) and GC content at the third synonym codon position (GC3s). Three types of plots including ENC-GC3s, parity rule 2 (PR2) and neutrality plots were employed to discover the factors determining CUB, and the frequency of hydrogen bonds and nucleotide were calculated to dissect changes in GC content at the 5′-end of the coding sequence. Our results showed little distinctness in CUB among the five species, with a reduction of hydrogen bonds content at the 5′-end (with similar changes in cytosines). In addition, optimal codons of Ipomoea aquatica ended with G or C, different from those of the other four species, which ended in A or T. These results may be useful for exploring the evolutionary relationships among this group, and for understanding the reasons for the variation among Ipomoea species. • The number of hydrogen bonds in each codon at the 5′-end of CDS both exhibited a decline trend among five Ipomoea species. • Natural selection plays a dominant role in the codon usage bias pattern of five Ipomoea species. • The optimal codons in Ipomoea aquatica ended with G or C, but other four species ended with A or T. [ABSTRACT FROM AUTHOR]