Weedy species of the genus Amaranthus, commonly referred to as pigweeds, have increased in frequency and severity over the past few years. Identification of these weeds is difficult because of similar morphological characteristics among species and variation within species. Studies were initiated to develop a molecular marker identification system utilizing restriction enzyme analysis of amplified ribosomal DNA (rDNA). A set of polymerase chain reaction (PCR) markers was developed to distinguish 10 weedy species of pigweeds. Restriction-site variation, utilizing five endonucleases, within the internal transcribed spacers (ITS) of the rDNA allowed for the positive identification of eight species and one pair of species. These markers will be useful for biological and ecological studies on the genus.