Green algal endophytes assigned to the genus Acrochaete were found within a population of Chondrus crispus Stackhouse (Rhodophyta) located at Lilstock Beach, Somerset, UK. Analysis of sequence data derived from the ribosomal DNA internal transcribed spacer 2 (ITS2) non-coding region of five clonal isolates of Acrochaete heteroclada, six of A. operculata and eight of A. viridis revealed that 31% of nucleotide positions were variable and suggested a close relationship with Enteromorpha and Ulva species. Acrochaete viridis and A. operculata were more closely related to each other and to Ulva/Enteromorpha than they were to A. heteroclada. This grouping is different from that inferred from morphological data, suggesting that A. viridis is more closely related to A. heteroclada than it is to A. operculata. Further morphological and molecular characterization of both fresh isolates and type specimens is required to resolve the current taxonomic confusion regarding these endophytic algae. There is sufficient sequence variability of the rDNA ITS2 between Acrochaete species to allow the design of species-specific molecular probes. Such probes will allow the detection of endophytic DNA in bulk nucleic acids extracted from the potential hosts, such as C. crispus. Our results suggest that inadvertent characterization of cloned endophyte DNA may be responsible for the misclassification of some sequences deposited in publicly accessible sequence databases.