The VQ motif-containing proteins play a vital role in various processes such as growth, resistance to biotic and abiotic stresses and development. However, there is currently no report on the VQ genes in sugarcane (Saccharum spp.). Herein, 78 VQ genes in Saccharum spontaneum were identified and classified into nine subgroups (I-IX) by comparative genomic analyses. Each subgroup had a similar structural and conservative motif. These VQ genes expanded mainly through whole-genome segmental duplication. The cis-regulatory elements (CREs) of the VQ genes were widely involved in stress responses, phytohormone responses and physiological regulation. The RNA-seq data showed that SsVQ gene expression patterns in 10 different samples, including different developmental stages, revealed distinct temporal and spatial patterns. A total of 23 SsVQ genes were expressed in all tissues, whereas 13 SsVQ genes were not expressed in any tissues. Sequence Read Archive (SRA) data showed that the majority of SsVQs responded to cold and drought stress. In addition, quantitative real-time PCR analysis showed that the SsVQs were variously expressed under salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA) and cold treatment. This study conducted a full-scale analysis of the VQ gene family in sugarcane, which could be beneficial for the functional characterization of sugarcane VQ genes and provide candidate genes for molecular resistance breeding in cultivated sugarcane in the future.