Six species of Trifolium (T. polymorphum Poir., T. riograndense Burkart, T. argentinense Speg., T. medium L., T. pratense L. and T. repens L.) were analyzed using inter-simple sequence repeats (ISSR) markers. Six selected primers generated 186 polymerase chain reaction (PCR) products exploring 112 loci in 34 genotypes analyzed with molecular sizes ranging from 200 to 1300 bp. These primers were able to discriminate among and within species, with the PCR products being on average 41.6% species-specific and 59.9% polymorphic at the within species level. Nuclear DNA content was determined by flow cytometry and revealed variation among species. The 1Cx genome size values were calculated and were found to range from 0.46 pg (T. pratense) to 0.96 pg (T. polymorphum). Genome size values of South American species were higher than those of Eurasiatic origin. The analyses of the molecular data grouped the six species in agreement with their geographical origin and clearly differentiate T. polymorphum from T. argentinense. The Eurasiatic group showed the highest average of species-specific bands (45.3%) and the South American group exhibited the highest amount of total bands (59.7). The highest level of intra-species polymorphisms was detected in T. argentinense(92.9%), followed by T. medium(89.5%).