Bacterial wilt caused by Curtobacterium flaccumfaciens pv. flaccumfaciens is among the diseases that affect Phaseolus vulgaris L. This disease has been frequently detected in bean fields and causes severe production losses in Brazil. The aim of this research was to examine the genetic diversity existing among twenty-four isolates of C. flaccumfaciens collected from their native and alternative host, and a collection of sixty strains belonging to four phytopathogenic pathovars preserved at the French Collection for Plant-associated Bacteria (CIRM-CFBP) by multilocus sequence analysis (MLSA) based on six housekeeping genes (atpD, dnaK, gyrB, ppK, recA and rpoB). A phylogenetic tree with the concatenated sequences of six genes showed high genetic diversity among the strains. For instance, strains belonging to C. f. pv. flaccumfaciens do not cluster together within the species. Similar results were obtained with a minimal MLSA scheme using gyrB and recA, which we propose for reliable identification at the species level of Curtobacterium isolates. No correlation was identified between phylogeny and pathogenicity in the Curtobacterium flaccumfaciens strains analyzed in this work. The specific primers CffFOR2 and CffREV4 designed by Tegli et al. (Letters in Applied Microbiology, 35(4), 331–337, 2002) to detect C. f. pv. flaccumfaciens in naturally infected bean seeds proved to be efficient for the detection of bean-pathogenic strains.