Mobile genetic elements (MGEs) sequester and mobilize antibiotic resistance genes across bacterial genomes. Efficient and reliable identification of such elements is necessary to follow resistance spreading. However, automated tools for MGE identification are missing. Tyrosine recombinase (YR) proteins drive MGE mobilization and could provide markers for MGE detection, but they constitute a diverse family also involved in housekeeping functions. Here, we conducted a comprehensive survey of YRs from bacterial, archaeal, and phage genomes and developed a sequence‐based classification system that dissects the characteristics of MGE‐borne YRs. We revealed that MGE‐related YRs evolved from non‐mobile YRs by acquisition of a regulatory arm‐binding domain that is essential for their mobility function. Based on these results, we further identified numerous unknown MGEs. This work provides a resource for comparative analysis and functional annotation of YRs and aids the development of computational tools for MGE annotation. Additionally, we reveal how YRs adapted to drive gene transfer across species and provide a tool to better characterize antibiotic resistance dissemination. SYNOPSIS A systematic resource for tyrosine recombinase annotation is presented. Comparative sequence analysis of the protein family enables the functional classification of these enzymes and the identification of mobile genetic elements in bacterial genomes. Phylogenetic analysis of the tyrosine recombinase protein family classifies its members into twenty subgroups. Members of the subgroups have a specific function, sequence features and host taxonomy. Tyrosine recombinases of mobile genetic elements carry an additional arm‐binding domain. Tyrosine recombinase classification enables the identification of new mobile genetic elements in bacterial genomes. A systematic resource for tyrosine recombinase annotation is presented. Comparative sequence analysis of the protein family enables the functional classification of these enzymes and the identification of mobile genetic elements in bacterial genomes.