Calcium plays a critical role in regulating abiotic-stress response in plant. Calcineurin B-like (CBL) proteins have been identified as calcium sensors in the calcium signaling pathway in Arabidopsis and rice. However, molecular mechanism underlying calcium signaling remains to be elucidated, especially in poplar, a model tree species with complete sequencing of the whole genome. Here, we have characterized 10 CBL candidate genes in the genome of Populus trichocarpa (PtCBLs) and presented a comparative genomics analysis of CBLs in poplar, rice and Arabidopsis, including gene structures, conserved motifs and phylogeny. Comparative genomics analyses showed that the family in poplar appears to be rather conserved in the size and structure, and the duplication events might significantly contribute to the amplification of this gene family. Furthermore, to elucidate the functions of CBL gene members respectively in regulating plant response to external stimuli, we presented a study of CBL family in Populus euphratica (PeCBLs), a mostly salt-and drought-tolerant Populus species. Nine cDNA clones of PeCBL members were isolated from Populus euphratica and a gene specific RT-PCR was performed to investigate their expression patterns under abiotic stress treatment, such as cold, drought, and salt. A transcript analysis of CBLs in Populus euphratica under abiotic stress suggests that seven CBL gene (PeCBL1, 2, 3, 4, 5, 9, and 10) members may play an important role in correspondence to specific external stimuli. Our data will provide important evidence for further studying the functional dissection of the CBL signaling network in plant.