The α-crystallin domain-containing (ACD-containing) gene family, which includes typical small heat shock proteins (sHSPs), is the most ubiquitous and diverse family of putative chaperones in all organisms, including eukaryotes and prokaryotes. In the present study, approximately 54-117 ACD-containing genes were identified in five penaeid shrimp species, yielding a significant expansion in comparison with other crustaceans (generally 6-20 ACD-containing genes). Unlike typical sHSPs, which contain a single ACD domain, the ACD-containing genes of penaeid shrimp contain additional ACD domains (3-7 domains, in general), thus having a larger molecular weight and a more complex 3D structure. As indicated by the RNA-seq and qRT-PCR results, the ACD-containing genes of penaeid shrimp showed a strong response to high temperatures. Furthermore, heterologous expression and citrate synthase assays of three representative ACD-containing genes confirmed that their chaperone activity could enhance the thermo-tolerance of E. coli and prevent the aggregation of substrate proteins at high temperatures. Compared with penaeid shrimp species with a relatively low thermo-tolerance (Fenneropenaeus chinensis and Marsupenaeus japonicus), the species with high thermo-tolerance (Litopenaeus vannamei and Fenneropenaeus indicus) contained more ACD-containing genes due to tandem duplication and exhibited biased expression levels under high temperatures. This can explain the divergent thermo-tolerance of different penaeid shrimp species. In conclusion, the ACD-containing genes in penaeid shrimp could be assigned as new chaperones and contribute to their divergent thermo-tolerance phenotypes and adaptations to the ecological environment.