Screening of large numbers of Acinetobacter spp. from activated sludge systems with Pyrolysis Mass Spectrometry (PyMS) showed that many did not cluster tightly with the currently described genomic species which have been obtained mainly from clinical sources. Selected isolates were then genotypically fingerprinted using their 16S-23S rDNA spacer region, and again the data revealed considerable differences in the genomic fingerprints of many of these activated sludge isolates to the predominantly clinical genomic species. In fact, few could be identified from them. The possibility that the current speciation within this genus is not adequate to encompass all these environmental isolates is addressed in relation to the methods used to study the population dynamics of Acinetobacter in activated sludge.