Porifera (sponges) are the most basal phylum of extant metazoans. To gain insight into sponge genome construction, cytogenetic analysis was performed for ten freshwater sponge species of six genera, using conventional Giemsa staining, chromosome banding, and fluorescence in-situ hybridization. The karyotypes were very similar among the ten species, exhibiting a diploid chromosome number of 2n=46 or 48, and usually consisted of microchromosomes with one or two pairs of large chromosomes. The 18S-28S rRNA genes were localized to a single pair of microchromosomes in two Ephydatia species. Hybridization signals of the telomere (TTAGGG)_n sequences were observed at the ends of metaphase chromosomes. The genome sizes of Ephydatia fluviatilis and Ephydatia muelleri were estimated by flow cytometric analysis as about 0.7 pg per diploid complement. These freshwater sponge species appear to represent a fairly homogeneous group with respect to karyotypes.