Glycogen-accumulating organisms (GAO) are thought to out-compete the polyphosphate-accumulating organisms (PAO) in activated sludge communities removing phosphate (P). Two GAO groups are currently recognized, the gammaproteobacterial Candidatus'Competibacter phosphatis', and the alphaproteobacterial Defluviicoccus vanus-related tetrad forming organisms (TFOs). Both are phylogenetically diverse based on their 16S rRNA sequences, with the latter currently considered to contain members falling into three distinct clusters. This paper identifies members of an additional fourth Defluviicoccus cluster from 16S rRNA gene clone library data obtained from a laboratory-scale activated sludge plant community removing P, and details FISH probes designed against them. Probe DF181A was designed to target a single sequence and DF181B designed against the remaining sequences in the cluster. Cells hybridizing with these probes in the biomass samples tested always appeared as either TFOs or in large clusters of small cocci. Members of the Defluviicoccus-related organisms were commonly found in full-scale wastewater treatments plants, sometimes as a dominant population.