The ability of sphingomonads in drinking water to cause community- and hospital-acquired opportunistic infections has raised the need to establish reproducible identification assays. In this study, a total of 129 isolates recovered from drinking water with yellow- to orange-pigmented colonies were distributed among 10 biotypes on the basis of colony morphology. Polymorphisms, based on the amplification and restriction digestion of the intergenic transcribed spacer (ITS) region within the 10 assigned biotypes and 18 ATCC reference strains, were used to investigate the ability of this approach to differentiate closely related sphingomonads. ITS size, which ranged between 400 and 1100 bp, did not vary enough among the different genera. However, 16 distinct banding patterns within the ATCC reference strains and 9 within the 10 biotypes were obtained through ITS restriction digestion, and the majority of the tested biotypes produced patterns similar to those generated by the ATCC strains. To our knowledge, this study is not only the first comprehensive record of the size of the ITS region in sphingomonads, it is also the first study that describes the use of ITS restriction digestion to subtype those isolates.