The presence of the foodborne pathogen Listeria monocytogenes on cold-smoked salmon is a major concern for the seafood industry. Understanding processing and postprocessing handling factors that affect the ability of this pathogen to grow on cold-smoked salmon is critical for developing effective control strategies. In this study, we investigated the effect of curing method and freeze-thawing of cold-smoked salmon on (i) physicochemical properties and (ii) subsequent growth of genetically diverse strains of L. monocytogenes (inoculated after freeze-thawing) and endogenous lactic acid bacteria. The majority of the measured physicochemical properties were unaffected by freezing and thawing. Overall, wet-cured cold-smoked salmon had higher pH, water activity, and moisture, as well as lower fat, water-phase salt, and phenolic content compared with dry-cured cold-smoked salmon. The curing method and freeze-thawing did not affect growth of endogenous lactic acid bacteria. Freeze-thawing cold-smoked salmon prior to inoculation led to pronounced growth of L. monocytogenes at 7°C. The increase in cell density between days 0 and 30 was significantly (P = 0.0078) greater for cold-smoked salmon that was frozen and thawed prior to inoculation compared with nonfrozen cold-smoked salmon. On dry-cured, freeze-thawed cold-smoked salmon, L. monocytogenes had a lag phase ranging from 3.7 ± 0.1 to 11.2 ± 1.4 days compared with salmon that was wet cured and freeze-thawed, on which L. monocytogenes began to grow within 24 h. Variation in growth among L. monocytogenes strains was also observed, indicating the significance of assessing multiple strains. Further efforts to understand the impact of processing and postprocessing handling steps of cold-smoked salmon on the growth of genetically diverse L. monocytogenes will contribute to improved challenge study designs and data. This, in turn, will likely lead to more reliable and unbiased risk assessments and control measures.