Watermelon (Citrullus lanatus L.), which is an economically important cucurbit crop that is cultivated worldwide, is vulnerable to various adverse environmental conditions. Small heat shock protein 20s (HSP20s) are the most abundant plant HSPs and they play important roles in various biotic and abiotic stress responses. However, they have not been systematically investigated in watermelon. In this study, we identified 44 watermelon HSP20 genes and analyzed their gene structures, conserved domains, phylogenetic relationships, chromosomal distributions, and expression profiles. All of the watermelon HSP20 proteins have a conserved the α-crystallin (ACD) domain. Half of the ClHSP20s arose through gene duplication events. Plant HSP20s were grouped into 18 subfamiles and a new subfamily, nucleo-cytoplasmic XIII (CXIII), was identified in this study. Numerous stress- and hormone-responsive cis-elements were detected in the putative promoter regions of the watermelon HSP20 genes. Different from that in other species, half of the watermelon HSP20s were repressed by heat stress. Plant HSP20s displayed diverse responses to different virus infections and most of the ClHSP20s were generally repressed by Cucumber green mottle mosaic virus (CGMMV). Some ClHSP20s exhibited similar transcriptional responses to abscisic acid, melatonin, and CGMMV. Subcellular localization analyses of six selected HSP20- green fluorescence protein fusion proteins revealed diverse subcellular targeting. Some ClHSP20 proteins were affected by CGMMV, as reflected by changes in the size, number, and distribution of fluorescent granules. These systematic analyses provide a foundation for elucidating the physiological functions and biological roles of the watermelon HSP20 gene family. [ABSTRACT FROM AUTHOR]