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Background: Burkholderia pseudomallei and B. mallei are closely related Category B Select Agents of bioterrorism and the causative agents of the diseases melioidosis and glanders, respectively. Rapid phage-based diagnostic tools would greatly benefit early recognition and treatment of these diseases. There is extensive strain-to-strain variation in B. pseudomallei genome content due in part to the presence or absence of integrated prophages. Several phages have previously been isolated from B. pseudomallei lysogens, for example ϕK96243, ϕ1026b and ϕ52237. Results: We have isolated a P2-like bacteriophage, ϕX216, which infects 78% of all B. pseudomallei strains tested. ϕX216 also infects B. mallei, but not other Burkholderia species, including the closely related B. thailandensis and B. oklahomensis. The nature of the ϕX216 host receptor remains unclear but evidence indicates that in B. mallei ϕX216 uses lipopolysaccharide O-antigen but a different receptor in B. pseudomallei. The 37,637 bp genome of ϕX216 encodes 47 predicted open reading frames and shares 99.8% pairwise identity and an identical strain host range with bacteriophage ϕ52237. Closely related P2-like prophages appear to be widely distributed among B. pseudomallei strains but both ϕX216 and ϕ52237 readily infect prophage carrying strains. Conclusions: The broad strain infectivity and high specificity for B. pseudomallei and B. mallei indicate that ϕX216 will provide a good platform for the development of phage-based diagnostics for these bacteria. [ABSTRACT FROM AUTHOR]