RFLP and sequences of ITS-rDNA of 45 populations of cyst-forming nematodes collected from different parts of Iran were analysed and identified as representatives of 21 species. Eight enzymes generated RFLP for all studied populations. Comparison of RFLP profiles and sequences of the ITS regions with published data confirmed the presence of Heterodera avenae, H. filipjevi, H. glycines, H. hordecalis, H. latipons, H. schachtii and H. trifolii in Iran. RFLP patterns and ITS sequences for H. elachista, H. turcomanica, H. mothi and C. cacti were obtained for the first time in this study. Heterodera humuli, H. goettingiana, H. fici, H. elachista, H. turcomanica and Cactodera cacti are recorded for the first time in Iran. These results correspond with morphological and morphometric identification of the populations. Several populations were not identified at the species level and are attributed to Heterodera sp.; some of these may correspond to new species. Twenty-one new sequences from Iranian cyst-forming nematodes and 36 known sequences were used for the phylogenetic analyses. The cyst-forming nematodes formed several clades corresponding to their morphological features. Heterodera mothi and H. elachista clustered with high support with other Cyperi group species and H. turcomanica formed a moderately to highly supported clade with the Humuli group.