Zooxanthellae, algal symbionts in divergent marine invertebrate hosts, are a genetically heterogeneous group. All species descriptions and most physiological and infectivity studies of zooxanthellae have been conducted using cultured material. However, few studies have attempted to quantify the representation of cultures isolated from cnidarians to the in hospite zooxanthella populations of the individual host or host species from which they were established. RFLPs of small subunit (18S) rDNA, internal transcribed spacer (ITS)-rDNA sequence data, and microsatellite analyses were conducted to assess the relatedness between cultured zooxanthellae and the in hospite populations of the individual host or host species from which they were isolated. RFLP data demonstrated that cultures may represent either the numerically dominant symbiont or ones present in lower number. ITS-rDNA sequences from zooxanthella cultures were disconcordant with ITS-rDNA sequences identified from in hospite zooxanthellae of the same host species, and microsatellites present in in hospite zooxanthella populations were absent from the corresponding cultures. Finally, reexamination of the literature revealed examples of zooxanthella cultures being nonrepresentative of in hospite populations. These data suggest that, in most cases, cultures are a subset of the original in hospite population. Factors such as failing to homogenize bulk cultures before transfer, growth medium used, and the picking of single motile cells may contribute to many zooxanthella cultures being nonrepresentative.