We conducted a candidate gene-based approach to search for genetic associations between 10 SNPs in PsCBF2 and 10 abiotic stress-related traits. The increasing incidence of abiotic stresses and the limitations of available treatments, particularly in trees, highlight the need to improve our understanding of the mechanisms of stress responses. In Arabidopsis, C-repeat binding factor 2 (CBF2) plays an important role in freezing tolerance and cold acclimation. Here, we isolated orthologs of CBF2 from five Populus species. Expression profiling revealed that the Populus CBF2s were preferentially induced in response to cold, with CBF2 transcript abundances ranging from 5.4- to 62-fold higher than in unstressed controls of the corresponding species. In addition, we used a candidate gene-based approach in Populus simonii Carr. to identify single nucleotide polymorphisms (SNPs) in PsCBF2 associated with physiological and biochemical traits. PsCBF2 showed high nucleotide diversity (π T = 0.00549, θ w = 0.01406) and low average linkage disequilibrium (r (2) = 0.061). Association studies in 528 individuals of an association population showed that nine SNPs (false discovery rate Q < 0.10) and one haplotype (Q < 0.10) were significantly associated with differences in four physiological and biochemical traits (P < 0.005), with each marker explaining 1.31-5.87 % of the total variance in the corresponding trait. PsCBF2 transcript levels differed significantly in abundance among genotypic classes for most of the significant SNPs. Identification of these significant associations will help reveal the molecular basis of physiological differences and provide a starting point for marker-assisted selection for traits involved in stress tolerance in P. simonii.