The availability of large volumes of genome sequence presents an unprecedented challenge for proteomics to characterize various protein motifs in order to determine their structure and function. Primary sequence alignment is often unable to accurately identify a given motif due to sequence divergence. However, with the aid of secondary structural prediction for analysis, it is feasible to explore protein motifs on a proteome-wide scale. Here we report the use of secondary structural alignment to characterize the SH2 domains of both conventional and divergent sequences and divide them into two groups, Src type and STAT type. In addition to the basic “abbba” structure, the Src type SH2 domain contains an extra b-strand, i.e., the bE or bE-bF motif. Whereas, the linker domain conjugated SH2 domain in STAT contains the aB’ motif instead. We combined the bB core motif sequence BLAST with secondary structural alignment to screen for SH2 domains in various eukaryotic model systems including Arabidopsis, Dictyostelium and Saccharomyces. Two novel genes carrying the linker-SH2 domain of STAT were discovered and subsequently cloned from Arabidopsis. These genes, designated as STAT-like factors (STATL), exist widely in vascular and nonvascular plants suggesting that STAT’s linker-SH2 domain was not acquired accidentally by Arabidopsis, rather that they co-evolved prior to the divergence of plants and animals. Using this approach, we expanded the number of putative SH2 domain-bearing genes in Dictyostelium and comparatively studied the secondary structural profiles of both typical and atypical SH2 domains. Our results indicate that the linker-SH2 domain of the transcription factor STAT is one of the most ancient and fully developed functional domains, which may have served as a template for the continuing evolution of the SH2 domain essential for phosphotyrosine signal transduction.