The categorization of genes by structural distinctions relevant to biological characteristics is very important for understanding of gene functions and predicting functional implications of uncharacterized genes. It was absolutely necessary to deploy an effective and efficient strategy to deal with the complexity of the large olfactomedin-like (OLF) gene family sharing sequence similarity but playing diversified roles in many important biological processes, as the simple highest-hit homology analysis gave incomprehensive results and led to inappropriate annotation for some uncharacterized OLF members. In light of evolutionary information that may facilitate the classification of the OLF family and proper association of novel OLF genes with characterized homologs, we performed phylogenetic analysis on all 116 OLF proteins currently available, including two novel members cloned by our group. The OLF family segregated into seven subfamilies and members with similar domain compositions or functional properties all fell into relevant subfamilies. Furthermore, our Northern blot analysis and previous studies revealed that the typical human OLF members in each subfamily exhibited tissue-specific expression patterns, which in turn supported the segregation of the OLF subfamilies with functional divergence. Interestingly, the phylogenetic tree topology for the OLF domains alone was almost identical with that of the full-length tree representing the unique phylogenetic feature of full-length OLF proteins and their particular domain compositions. Moreover, each of the major functional domains of OLF proteins kept the same phylogenetic feature in defining similar topology of the tree. It indicates that the OLF domain and the various domains in flanking non-OLF regions have coevolved and are likely to be functionally interdependent. Expanded by a plausible gene duplication and domain couplings scenario, the OLF family comprises seven evolutionarily and functionally distinct subfamilies, in which each member shares similar structural and functional characteristics including the composition of coevolved and interdependent domains. The phylogenetically classified and preliminarily assessed subfamily framework may greatly facilitate the studying on the OLF proteins. Furthermore, it also demonstrated a feasible and reliable strategy to categorize novel genes and predict the functional implications of uncharacterized proteins based on the comprehensive phylogenetic classification of the subfamilies and their relevance to preliminary functional characteristics.