Conjugative plasmids are important drivers of bacterial evolution. Most plasmids lack genes for conjugation and characterized origins of transfer (oriT), which has hampered our understanding of plasmid mobility. Here we used bioinformatic analyses to characterize occurrences of known oriT families across 38,057 plasmids, confirming that most conjugative and mobilizable plasmids lack identifiable oriTs. Recognizable oriT sequences tend to be intergenic, upstream of relaxase genes and specifically associated with relaxase types. We used these criteria to develop a computational method to search for and identify 21 additional families of oriT-containing sequences in plasmids from the pathogens Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii. Sequence analyses found 3,072 occurrences of these oriT-containing sequences across 2,976 plasmids, many of which encoded antimicrobial resistance genes. Six candidate oriT-containing sequences were validated experimentally and were shown to facilitate conjugation in E. coli. These findings expand our understanding of plasmid mobility.