Heat shock proteins 70 have triggered a remarkable large body of research in various fishes; however, no genome-wide identification and expression analysis has been performed on the Hsp70 gene family of Boleophthalmus pectinirostris. In this study, we identified 20 Hsp70 genes within the genome of B. pectinirostris and provided insights into their response to high environmental ammonia (HEA) stress. Positive selection on stress response genes and expansion of hspa1a and hspa1a-like genes might be related to terrestrial adaptations in this species. The expression patterns of the Hsp70 gene family in the gill and liver of B. pectinirostris under HEA stress were studied by examining transcriptome data. The results showed that most Hsp70 genes were downregulated after high concentration ammonia exposure. The downregulation may be related to the hypoxic condition of the tissues.