Macroalgae of the order Laminariales (kelps) are important marine coastal primary producers. Important factors influencing their distribution include light and temperature; despite kelp prime importance for ecosystem function, the molecular basis of these limitations is still poorly understood. In order to fill this gap we constructed a comprehensive cDNA library from RNA sampled under various light and temperature regimes as a basis for future studies about the mechanisms and pathways involved in acclimation to light and temperature stress in Saccharina latissima. A total of 400,503 ESTs was assembled into 28,803 contigs. We were able to assign putative functions or orthology relationships to more than 10,000 contigs either by BLASTx, Interpro protein motif annotation, or Gene Ontology. The most frequent Interpro protein domains found in the cDNA library were the protein kinase-like domain, serine/threonine-protein kinase-like domain, NAD(P)-binding and thioredoxin-like fold domain. Enzyme code (EC) annotation yielded in the attribution of 480 contigs, providing a total of 625 ECs, which could be mapped to 85 biochemical pathways. Comparative genomics of Saccharina latissima and Ectocarpus siliculosus revealed genome coverage of approximately 70% of our cDNA library, under the assumption of similar gene numbers in both species. GO term occurrence in S. latissima and E. siliculosus showed a similar distribution pattern among the root ontologies biological process, molecular function and cellular component. Comparative protein domain annotation of S. latissima und E. siliculosus showed that, probably due to the chosen stress conditions, the domains “thioredoxin fold”, “thioredoxin-like fold”, “heat shock protein 70”, and “bromoperoxidase/chloroperoxidase C-terminal” are over-represented in the cDNA library.