Glycoside Hydrolase Family 16 (GH16) comprises a large and taxonomically diverse family of glycosidases and transglycosidases that adopt a common beta-jelly-roll fold and are active on a range of terrestrial and marine polysaccharides. Presently, broadly insightful sequence-function correlations in GH16 are hindered by a lack of a systematic subfamily structure. To fill this gap, we have used a highly scalable protein Sequence Similarity Network (SSN) analysis to delineate nearly 23,000 GH16 sequences into 23 robust subfamilies, which are strongly supported by Hidden Markov Model (HMM) and Maximum Likelihood (ML) molecular phylogenetic analyses. Subsequent evaluation of over 40 experimental three-dimensional structures has highlighted key tertiary structural differences, predominantly manifested in active-site loops, which dictate substrate specificity across the GH16 evolutionary landscape. As for other large GH families (i.e. GH5, GH13, and GH43), this new subfamily classification provides a roadmap for functional glycogenomics that will guide future bioinformatics and experimental structure-function analyses. The GH16 subfamily classification is publicly available in the CAZy database via URL www.cazy.org/GH16.html. The SSN workflow used here is available via URL https://github.com/ahvdk/SSNpipe/.