Sole carbon source utilization (SCSU) patterns and phospholipid fatty acid (PLFA) profiles were compared with respect to their potential to characterize root-inhabiting microbial communities of hydroponically grown crops. Sweet pepper (Capsicum annum cv. Evident), lettuce (Lactuca sativa cv. Grand Rapids), and four different cultivars of tomato (Lycopersicon esculentum cvs. Gitana, Armada, Aromata, and Elin) were grown in 1-L black plastic beakers placed in a cultivation chamber with artificial light. In addition to the harvest of the plants after 6 weeks, plants of one tomato cultivar, cv. Gitana, were also harvested after 4 and 8 weeks. The cultivation in this study was performed twice. Principal component analysis was used to analyze the data. Both characterization methods had the ability to discriminate between the root microflora of different plant species, cultivars, and one tomato cultivar at different ages. Differences in both SCSU patterns and PLFA profiles were larger between plant species than between cultivars, but for both methods the largest differences were between the two cultivations. Still, the differences between treatments were always due to differences in the same PLFAs in both cultivations. This was not the case for the SCSU patterns when different plant ages were studied. Furthermore, PLFA profiles showed less variation between replicates than did SCSU patterns. This larger variation observed among the SCSU data indicates that PLFA may be more useful to detect changes in the root microflora of hydroponically grown crops than the SCSU technique.