WW domains are protein modules that mediate protein-protein interactions through recognition of proline-rich peptide motifs and phosphorylated serine/threonine-proline sites. WW domains are found in many different structural and signaling proteins that are involved in a variety of cellular processes. WW domain-containing proteins (WWCPs) and complexes have been implicated in major human diseases including cancer as well as in major signaling cascades such as the Hippo tumor suppressor pathway, making them targets for new diagnostics and therapeutics. There are a number of reports about the WWCPs in different species, but systematic analysis of the WWCP genes and its ligands is still lacking in silkworm and the other organisms. In this study, WWCP genes and PY motif-containing proteins have been identified and analyzed in 56 species including silkworm. Whole-genome screening of B. mori identified thirty-three proteins with thirty-nine WW domains located on thirteen chromosomes. In the 39 silkworm WW domains, 15 domains belong to the Group I WW domain; 14 domains were in Group II/III, 9 domains derived from 8 silkworm WWCPs could not be classified into any group, and Group IV contains only one WW domain. Based on gene annotation, silkworm WWCP genes have functions in multi-biology processes. A detailed list of WWCPs from the other 55 species was sorted in this work. In 14,623 silkworm predicted proteins, nearly 18 % contained PY motif, nearly 30 % contained various motifs totally that could be recognized by WW domains. Gene Ontology and KEGG analysis revealed that dozens of WW domain-binding proteins are involved in Wnt, Hedgehog, Notch, mTOR, EGF and Jak-STAT signaling pathway. Tissue expression patterns of WWCP genes and potential WWCP-binding protein genes on the third day of the fifth instar (L5D3) were examined by microarray analysis. Tissue expression profile analysis found that several WWCP genes and poly-proline or PY motif-containing protein genes took tissue- or gender-dependent expression manner in silkworms. We further analyzed WWCPs and PY motif-containing proteins in representative organisms of invertebrates and vertebrates. The results showed that there are no less than 16 and up to 29 WWCPs in insects, the average is 22. The number of WW domains in insects is no less than 19, and up to 47, the average is 36. In vertebrates, excluding the Hydrobiontes, the number of WWCPs is no less than 34 and up to 49, the average is 43. The number of WW domains in vertebrates is no less than 56 and up to 85, the average is 73. Phylogenetic analysis revealed that most homologous genes of the WWCP subfamily in vertebrates were duplicated during evolution and functions diverged. Nearly 1,000 PY motif-containing protein genes were found in insect genomes and nearly 2,000 genes in vertebrates. The different distributions of WWCP genes and PY motif-containing protein genes in different species revealed a possible positive correlation with organism complexity. In conclusion, this comprehensive bio-information analysis of WWCPs and its binding ligands would provide rich fundamental knowledge and useful information for further exploration of the function of the WW domain-containing proteins not only in silkworm, but also in other species.