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Background  Discerning genes crucial to antimicrobial resistance (AMR) mechanisms is becoming more and more important to accurately and swiftly identify AMR pathogenic strains. Pangenome-wide association studies (e.g. Scoary) identified numerous putative AMR genes. However, only a tiny proportion of the putative resistance genes are annotated by AMR databases or Gene Ontology. In addition, many putative resistance genes are of unknown function (termed hypothetical proteins). An annotation tool is crucially needed in order to reveal the functional organization of the resistome and expand our knowledge of the AMR gene repertoire.    Results  We developed an approach (PangenomeNet) for building co-functional networks from pan-genomes to infer functions for hypothetical genes. Using  Escherichia coli  as an example, we demonstrated that it is possible to build co-functional network from its pan-genome using co-inheritance, domain-sharing, and protein–protein-interaction information. The investigation of the network revealed that it fits the characteristics of biological networks and can be used for functional inferences. The subgraph consisting of putative meropenem resistance genes consists of clusters of stress response genes and resistance gene acquisition pathways. Resistome subgraphs also demonstrate drug-specific AMR genes such as beta-lactamase, as well as functional roles shared among multiple classes of drugs, mostly in the stress-related pathways.    Conclusions  By demonstrating the idea of pan-genome-based co-functional network on the  E. coli  species, we showed that the network can infer functional roles of the genes, including those without functional annotations, and provides holistic views on the putative antimicrobial resistomes. We hope that the pan-genome network idea can help formulate hypothesis for targeted experimental works.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12859-021-04459-z.

PangenomeNet: a pan-genome-based network reveals functional modules on antimicrobial resistome for Escherichia coli strains