Phototrophic anoxygenic purple bacteria play a key role in many aquatic ecosystems by oxidizing sulfur compounds and low-molecular-weight organic compounds using light as energy source. In this study, molecular methods based upon pufM gene (photosynthetic unit forming gene) were compared with culture-dependent methods to investigate anoxygenic purple phototrophic communities in sediments of an eutrophic brackish lagoon. Thirteen strains, belonging to eight different genera of purple phototrophic bacteria were isolated with a large dominance of the metabolically versatile purple non-sulfur bacteria (eight strains), some purple sulfur bacteria (three strains) and two strains belonging to the Roseobacter clade (aerobic phototrophs). The pufM genes amplified from the isolated strains were not detected by the molecular methods [terminal-restriction fragment length polymorphism (T-RFLP)] applied on in situ communities. An environmental clone library of the pufM gene was thus constructed from sediment samples. The results showed that most of the clones probably corresponded to aerobic phototrophic bacteria. Our results demonstrate that the culture-dependent techniques remain the best experimental approach for determining the diversity of phototrophic purple non-sulfur bacteria whereas the molecular approach clearly illustrated the abundance of organisms related to the Roseobacter clade in these eutrophic sediments.