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Immune defense functions of silver carp (Hypophthalmichthys molitrix) and bighead carp (Hypophthalmichthys nobilis) have shown obvious evolutionary divergence. MiRNAs participate in the fine regulation of immune function. However, the evolutionary adaptation of miRNAs in the regulation of immune defense function is still poorly understood in silver carp and bighead carp. Here, small RNA libraries were constructed from the spleen tissue of one-year-old and three-year-old healthy silver carp and bighead carp, 424 and 422 known conserved miRNAs were respectively identified from the spleen of silver carp and bighead carp by bioinformatic analysis, which 398 were shared between the two species. These conserved miRNAs showed highly similar expression patterns between silver carp and bighead carp, but the abundance in spleen varied greatly in different species. Family analysis showed that miRNA families including mir-8, mir-7, mir-23, mir-338, mir-30, mir-27, mir-221, mir-19, mir-181, mir-17, mir-15, mir-148, mir-130, mir-10 and let-7 were the main miRNAs in the spleen of silver carp and bighead carp. 27 and 51 significant differentially expressed (SDE) miRNAs were identified from silver carp and bighead carp, respectively. Evolution analysis for the predicted target genes of SDE-miRNAs showed that ten biological processes such as blood coagulation, cell adhesion mediated by integrin and adaptive immune response were positively selected. In addition, immune genes including TLR3 , NFATC3 , MALT1 , B2M , GILT and MHCII were positively selected only in silver carp, and they were specifically targeted by the SDE-miRNAs including miR-9-5p, miR-196a-5p, miR-375, miR-122, miR-722, miR-132-3p, miR-727-5p, miR-724, miR-19d-5p and miR-138-5p, respectively. PLA2G4 in Fc epsilon RI signaling pathway was positively selected only in bighead carp and was specifically targeted by the SDE-miRNAs including miR-222b, miR-22b-5p, miR-15c, miR-146a, miR-125c-3p, miR-221-5p, miR-2188-5p, miR-142a-3p, miR-212, miR-138-5p and miR-15b-5p. In particular, SDE-miRNAs such as miR-144-3p, miR-2188-3p, miR-731, miR-363-3p and miR-218b could simultaneously target multiple evolutionarily differentiated immune-related genes. These results indicated that in the spleen of silver carp and bighead carp, conserved miRNAs have obvious evolutionary adaptations in the regulation of immune defense function. The results of this study can provide valuable resources for further revealing themechanism of miRNA in the formation of resistance traits evolution between silver carp and bighead carp. • We described characteristics of conserved microRNAome in spleen of silver carp and bighead carp. • We found highly similar expression patterns of splenic conserved miRNAs between the two species. • We identified 27 and 51 SDE miRNA from the spleen of silver carp and bighead carp, respectively. • We found significant evolutionary adaptation of SDE miRNA in regulation of spleen immune function. • We found 7 key immune gene and their interaction miRNA in regulation of evolutionary divergence. [ABSTRACT FROM AUTHOR]