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The cell wall plays an important role in responses to various stresses. The cellulose synthase-like gene (<i>Csl</i>) family has been reported to be involved in the biosynthesis of the hemicellulose backbone. However, little information is available on their involvement in plant tolerance to low-temperature (LT) stress. In this study, a total of 42 <i>Csls</i> were identified in <i>Musa acuminata</i> and clustered into six subfamilies (<i>CslA</i>, <i>CslC</i>, <i>CslD</i>, <i>CslE</i>, <i>CslG</i>, and <i>CslH</i>) according to phylogenetic relationships. The genomic features of <i>MaCsl</i> genes were characterized to identify gene structures, conserved motifs and the distribution among chromosomes. A phylogenetic tree was constructed to show the diversity in these genes. Different changes in hemicellulose content between chilling-tolerant and chilling-sensitive banana cultivars under LT were observed, suggesting that certain types of hemicellulose are involved in LT stress tolerance in banana. Thus, the expression patterns of <i>MaCsl</i> genes in both cultivars after LT treatment were investigated by RNA sequencing (RNA-Seq) technique followed by quantitative real-time PCR (qPCR) validation. The results indicated that <i>MaCslA4/12</i>, <i>MaCslD4</i> and <i>MaCslE2</i> are promising candidates determining the chilling tolerance of banana. Our results provide the first genome-wide characterization of the <i>MaCsls</i> in banana, and open the door for further functional studies.