Interstitial polychaetes of the genus Ophryotrocha are very small, progenetic, and morphologically very similar. These worms have been widely used in evolutionary biology and sexuality studies. To have a better insight into the karyological evolution of this genus, we measured the total karyotypic length and the 2C nuclear DNA content of the nine best-known species of this genus. No interspecific differences were observed in karyotypic lengths, apart from that of O. gracilis, which was significantly greater than the karyotypic length of five of the nine species. The genome size (i.e., 1C DNA content calculated from 2C DNA content) in eight of the nine species is about 0.4 pg, irrespective of the chromosome number. A group of four gonochoric and morphologically indistinguishable species, with 2n = 6 metacentric chromosomes, appears to be heterogeneous with regard to its DNA content, because one of the species, O. macrovifera, has a genome twice the size of that of the other three species. A hermaphroditic species, O. hartmanni, has a genome three times that size. No correlation has been observed between genome size and body size, egg cell diameter, or time interval from egg fertilization to sexual maturity. The basic genome size of 0.4 pg is among the lowest recorded in invertebrates. Hypotheses about selective pressures that maintain such a low amount of nuclear DNA in this genus are discussed.