The roles of ‘silent’ synonymous mutations for adapting to stressful thermal environments are of fundamental biological and ecological interests but poorly understood. To study whether synonymous mutations influence thermal adaptation at specific microhabitats, a genome-wide genotype-phenotype association analysis was carried out in the black mussels Mytilisepta virgata. A synonymous mutation of Ubiquitin-specific Peptidase 15 (MvUSP15) was significantly associated with the physiological upper thermal limit. The individuals carrying GG genotype (the G-type) at the mutant locus possessed significantly lower heat tolerance compared to the individuals carrying GA and AA genotype (the A-type). When heated to sublethal temperature, the G-type exhibited higher inter-individual variations in MvUSP15 expression, especially for the mussels on the sun-exposed microhabitats. Taken together, a synonymous mutation in MvUSP15 can affect the gene expression profile and interact with microhabitat heterogeneity to influence thermal resistance. This integrative study sheds light on the ecological importance of adaptive synonymous mutations as an underappreciated genetic buffer against heat stress and emphasizes the importance of integrative studies at a microhabitat scale for evaluating and predicting the impacts of climate change.