Using wheat ditelosomic lines and in situ hybridization of biotin-labelled DNA probes, 18 restriction fragment length polymorphism (RFLP) markers were physically located on homoeologous groups 1 and 3 chromosomes of wheat. Most of the markers hybridized to chromosome arms in a physical order concordant with the genetic maps. A majority of the markers studied were clustered in non-C-banded, distal euchromatic areas, indicating the presence of recombination hot spots and cold spots in those regions. However, on IBS the markers were well dispersed, which could be due to the abundance of heterochromatin throughout the arm. An inversion between Xpsr653 and Xpsr953 was observed on 1AL. One new Xpsr688 locus, approximately 20-26% from the centromere, was found on 1AS and 1BS. The physical location of Xpsr170 on group 3 chromosomes probably represents an alternative to the loci on the genetic map. Finally, Xpsr313 was mapped to two physical loci on IDL. Five markers were located to bins consistent with the deletion-based physical maps.