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Summary: Manila clam, Ruditapes philippinarum, is an economically important marine bivalve species. Y‐box proteins are members of the cold shock proteins family and highly conserved from bacteria to humans. In this study, a novel Y‐box gene (Rpybx) was cloned from the Manila clam and gene expression profiling was performed on three shell color strains (white, zebra and white zebra) and two wild populations (Southern and Northern) of R. philippinarum. The complete ORF length of Rpybx is 1367 bp, encoding 253 amino acids residues. Based on the amino acid sequence analysis and phylogenetic analysis, the Rpybx gene was identified as a member of the invertebrate Y‐box proteins family. Rpybx has a similar tertiary structure to human Y‐box protein YB‐1. The Rpybx mRNA levels were analyzed by qPCR under acute and gradually varied cold stress. Under acute low‐temperature stress, the expression of Rpybx mRNA in gills and hepatopancreas was significantly increased in all selected strains and populations (P < 0.05). The Northern population showed the lowest relative expression level of Rpybx. The expressions of Rpybx were greatly upregulated in gills and hepatopancreas of different stains and populations at 5 or −2°C under gradually varied temperature stress (P < 0.05). The results shed light on the biological function of the Rpybx gene in defending against low‐temperature challenge and further exploring the molecular mechanism of cold tolerance and resistance in R. philippinarum. [ABSTRACT FROM AUTHOR]