Ribosomal DNA (rDNA) spacer length polymorphism was studied in foxtail millet (Setaria italica ssp. italica) landraces from Pakistan and Afghanistan and in its wild ancestor (S. italica ssp. viridis) from Pakistan by PCR-based methods. Sequence polymorphism was also investigated for accessions selected based on the observed length polymorphism. The PCR-based length polymorphism and sequence polymorphism of rDNA intergenic spacer (IGS) clearly demonstrated genetic differentiation between cultivated and wild forms in the region. Genetic differentiation was observed between different areas to some extent in the cultivated form, and between different regions in the wild form of northern Pakistan. Based on the results, we discuss the genetic differentiation of foxtail millet and wild ancestor in this region and possible utility of rDNA markers to trace the dispersal of this crop in the region.