This study reports the first description and molecular characterization of naturally occurring, non-bioluminescent strains ofVibrio fischeri. These ‘dark’V. fischeristrains remained non-bioluminescent even after treatment with both autoinducer and aldehyde, substrate additions that typically maximize light production in dim strains of luminous bacteria. Surprisingly, the entireluxlocus (eight genes) was absent in over 97% of these darkV. fischeristrains. Although these strains were all collected from a Massachusetts (USA) estuary in 2007, phylogenetic reconstructions allowed us to reject the hypothesis that these newly described non-bioluminescent strains exhibit monophyly within theV. fischericlade. These dark strains exhibited a competitive disadvantage against native bioluminescent strains when colonizing the light organ of the modelV. fischerihost, the Hawaiian bobtail squidEuprymna scolopes. Significantly, we believe that the data collected in this study may suggest the first observation of a functional, parallel locus-deletion event among independent lineages of a non-pathogenic bacterial species.