A data-independent acquisition assay library was generated for the liver of threespine sticklebacks to evaluate alterations in protein abundance and functional enrichment of molecular pathways following either a chronic warm (25°C) or cold (7°C) three-week temperature challenge in two estuarine populations. The assay library was created from a data-dependent acquisition (DDA) based raw spectral library that was filtered to remove low quality or ambiguous peptides. Functional enrichment analyses using STRING aided in identifying larger networks and domains that were significantly enriched in the different groups. The Big Lagoon population (BL) had a stronger response to both temperatures than the Klamath River population (KL). At 7°C, BL showed alterations in protein homeostasis that likely fueled a higher demand for energy, but both populations appeared to successfully acclimate to this temperature. The warm temperature acclimation saw major increases in proteins involved in chromatin structure, including a variety of histones, and transcription, while there were decreases in proteins related to translation and fatty acid metabolism. Functional enrichment analyses uncovered differences in metabolism and highlighted altered molecular pathways that only had a few representative proteins reach significance from the individual quantification analyses. This data set represents the unique proteomic signatures of two populations of threespine sticklebacks and helps elucidate general themes as well as highlight population differences in acclimating to chronic temperature stress. The synchronous expression patterns of numerous proteins gives better context and specificity to a particular stressor and helps identify the strategies and mechanisms utilized to overcome changes in the environment.