The entire internal transcribed spacer (ITS) region, including the 5.8S subunit of the nuclear ribosomal DNA (rDNA), was sequenced by direct double-stranded sequencing of polymerase chain reaction (PCR) amplified fragments. The study included 40 Sporobolus (Family Poaceae, subfamily Chloridoideae) seed collections from 14 putative species (all 11 species from the S. indicus complex and three Australian native species). These sequences, along with those from two out-group species [ Pennisetum alopecuroides (L.) Spreng. and Heteropogon contortus (L.) P. Beauv. ex Roemer & Schultes, Poaceae, subfamily Panicoideae], were analysed by the parsimony method (PAUP; version 4.0b4a) to infer phylogenetic relationships among these species. The length of the ITS1, 5.8S subunit and ITS2 region were 222, 164 and 218 base pairs (bp), respectively, in all species of the S. indicus complex, except for the ITS2 region of S. diandrus P.Beauv. individuals, which was 217 bp long. Of the 624 characters included in the analysis, 245 (39.3%) of the 330 variable sites contained potential phylogenetic information. Differences in sequences among the members of the S. pyramidalis P.Beauv., S. natalensis (Steud.) Dur & Schinz and S. jacquemontii Kunth. collections were 0%, while differences ranged from 0 to 2% between these and other species of the complex. Similarly, differences in sequences among collections of S. laxus B.K.Simon, S. sessilis B.K.Simon, S. elongatus R.Br. and S. creber De Nardi were 0%, compared with differences of 1–2% between these four species and the rest of the complex. When comparing S. fertilis (Steud.) Clayton and S. africanus (Poir.) Robyns & Tourney, differences between collections ranged from 0 to 1%. Parsimony analysis grouped all 11 species of the S. indicus complex together, indicating a monophyletic origin. For the entire data set, pair-wise distances among members of the S. indicus complex varied from 0.00 to 1.58%, compared with a range of 20.08–21.44% among species in the complex and the Australian native species studied. A strict consensus phylogenetic tree separated 11 species of the S. indicus complex into five major clades. The phylogeny, based on ITS sequences, was found to be congruent with an earlier study on the taxonomic relationship of the weedy Sporobolus grasses revealed from random amplified polymorphic DNA (RAPD). However, this cladistic analysis of the complex was not in agreement with that created on past morphological analyses and therefore gives a new insight into the phylogeny of the S. indicus complex.