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Mycobacteriophage SWU1 is a newly isolated phage from soil sample collected in Sichuan province, China using Mycobacterium smegmatis mc²155 as host. Plaque, phage morphology and one-step growth curve were characterized. The complete genomic sequence of phage SWU1 was determined by shotgun sequencing. The ends of SWU1 were determined. Structural proteins of SWU1 were analyzed by NanoLC-ESI-MS/MS. Seven ORFs were identified as structural protein encoded by SWU1 genome. The genetic basis underlying the SWU1 plaque was explored using comparative genomics. Prophages homologous to SWU1 were identified in two pathogens, Segniliparus rugosus ATCC BAA-974 and Mycobacterium rhodesiae JS60. Genus Segniliparus is a member of the order Corynebacteriales. To our knowledge, this is the first report of Mycobacterium prophages in different genera.