Nodulin genes are involved in nodulation in leguminous species. Nodulin-like genes are also found in non-nodulating species, playing important roles during plant development. The Medicago truncatula NODULIN 21 (MtN21) family is one member of the nodulin-like gene family. However, only a few MtN21-like genes have been characterized. We compared MtN21-like genes in cotton with MtN21-like genes in Arabidopsis by bioinformatics methods. MtN21-like genes in cotton are homologous to those in Arabidopsis. MtN21-like proteins in cotton and Arabidopsis both have the same transmembrane domains, such as EamA and PLN00411. In cotton, the isoelectric points are predicted to be lower in MtN21-like proteins containing only the PLN00411 domain than those of MtN21-like proteins containing the EamA domain. The MtN21-like proteins are predicted to localize to the plasma membrane, tonoplast, chloroplast or nucleus in cotton. MtN21-like proteins in cotton have phosphorylation sites in the cytoplasmic portion of membrane. MtN21-like proteins in cotton may be transmembrane proteins with transporter activities. They may play roles in development and defense against pathogens in cotton.