Twenty cypress accessions were tested for freezing tolerance. After freezing to −15°C, differences among cypress accessions were tested by measuring electrolyte leakage and chlorophyll fluorescence. Based on these data, cypress accessions showing contrasting freezing tolerance were subjected to transcript profiling of candidate genes upon the development of cold hardening, with the ultimate goal of providing a scientific basis for selecting/breeding cypress genotypes with higher tolerance to low temperature. Nine different cypress genes were selected: a heat shock protein, a putative chaperonin, a chlorophyll-binding protein, a serine/threonine protein kinase, a putative exonuclease, a dehydrin, and three senescence-associated proteins. Transcript levels of these genes were profiled during cold hardening under controlled conditions using real-time reverse-transcription-polymerase chain reaction. While the genes showed regulation patterns common to both cypress accessions, in the case of chaperonin, exonuclease, and some senescence-associated proteins, clonal differences in gene regulation were found. The potential relationship of these differences with cold tolerance is discussed.