Updated FTP location. Spider mites are a group of arachnids belonging in the family Tetranychidae, known to produce nano-scale silk fibers characterized by high Young’s modulus. Silk fibroin gene of spider mites has been computationally suggested through genomic analysis of Tetranychus urticae, but it is yet to be confirmed by proteomic evidences. In this work, we sequenced and assembled the transcriptome from two genera of spider mites, Tetranychus kanzawai and Panonychus citri, and combined with the silk proteomics of T. urticae and P. citri to characterize the fibroin genes through comparative genomics and multi-omics analysis. As a result, two fibroins were identified, that are different genes from the previous computational prediction. Amino acid composition and secondary structure suggests similarity to aciniform or cylindrical spidroins of spider silk, which partly mirrors their mechanical property exhibiting high Young’s modulus. Availability of full-length fibroin sequences of spider mites facilitates the study of the evolution of silk genes that emerged in multiple lineages sometimes in convergent manner, and in the industrial application of artificial protein fibers through the study of the amino acid sequence and the resulting mechanical properties of these silks.