Cold poses major physiological challenges to plants, especially long-lived trees. In trees occurring along variable temperature clines, the expected direction and consequences of selection on cold acclimation ability and freezing tolerance are not straightforward. Here we estimated selection in cold acclimation genes at two evolutionary timescales in all seven species of the American live oaks (Quercus subsection Virentes). Two cold response candidate genes were chosen: ICE1, a key gene in the cold acclimation pathway, and HOS1, which modulates cold response by negatively regulating ICE1. Two housekeeping genes, GAPDB and CHR11, were also analyzed. At the shallow evolutionary timescale, we demonstrate that HOS1 experienced recent balancing selection in the two most broadly distributed species, Q. virginiana and Q. oleoides. At a deeper evolutionary scale, a codon-based model of evolution revealed the signature of negative selection in ICE1. In contrast, three positively selected codons have been identified in HOS1, possibly a signature of the diversification of Virentes into warmer climates from a freezing adapted lineage of oaks. Our findings indicate that evolution has favored diversity in cold tolerance modulation through balancing selection in HOS1 while maintaining core cold acclimation ability, as evidenced by purifying selection in ICE1.