Choline monooxygenase (CMO) is a key enzyme in the synthesis of betaine in higher plants and plays an important role in the physiological process of plant resistance to stress. A CMO gene was identified in banana A and B reference genomes in banana gene database, and the biological information analysis of MaCMO and MbCMO genome sequences showed that MbCMO might be formed by a gene encoding a banana O-fucosyltransferase family protein and MbCMO gene in tandem. The CMO gene coding sequences of Zhanjiang AA (ZJ; AA genotype), Baxijiao (BX; AAA genotype), Guangdong Dajiao (GD; AAB genotype) and Jinfen (JF; ABB genotype) were cloned and compared, it was found that ZJ and BX only contained CMO-A, GD and JF both contained CMO-A, CMO-B1 and CMO-B2, and JF also contained CMO-H. Codon usage characteristics showed that there were 23 common high frequency codons among the four banana CMO gene, and the codons CUU and CCG were the most biased and the weakest codons, respectively. The physicochemical properties of CMO-A protein and CMO-H protein showed that the minimum number of amino acids was 425, the maximum number of amino acids was 470, and the secondary structure of CMO-B1 protein was the most complex. The molecular weight of CMO-B2 protein was 52.02 kDa, and the molecular weight of CMO-A protein was 47.48 kDa. All the four CMO proteins were acidic proteins, which did not have a transmembrane structure and were hydrophilic proteins. Subcellular localization prediction showed that all four CMO were localized in chloroplasts. In terms of evolution, plant CMO had obvious branches in monodicotyledonous plants during evolution, and the CMO-A, CMO-H, CMO-B1 and CMO-B2 proteins of banana were more closely related to other monocotyledonous plant CMO proteins. The results of RT-qPCR showed that CMO expression was up-regulated in the four kinds of banana roots in the early stage of osmotic stress, and the expression levels of ZJ and BX CMO in homozygous A genome were higher than those of GD and JF CMO in heterozygous A and B genomes. CMO expression in the four kinds of banana leaves was down-regulated at the early stage of osmotic stress. The expression levels of homozygous ZJ and BX CMO in A genome were up-regulated at the late stage of osmotic stress, while the expression levels of heterozygous GD and JF CMO in A and B genomes peaked at 10 days and then down-regulated again at 15 days later. It was significantly lower than that of ZJ and BX CMO. This study revealed the differences of CMO genes between the A and B genomes of bananas and the expression patterns of CMO genes in different genotypes of bananas under osmotic stress, which would lay a foundation for further research on the biological functions of CMO genes in the A and B genomes of bananas, especially the relationship between CMO genes derived from different genomes and the ability of banana to resist osmotic stress. It would provide a reference for improving the stress resistance of banana by genetic engineering.