Five random amplified polymorphic DNA markers, two in coupling (OPAH5545C, and OPBA12314C) and three in repulsion phase (UBC285158R, OPC2441R, and OPB17451R), closely linked to Rsp genes conferring resistance to Septoria speckled leaf blotch (SSLB), were identified using bulked segregant analysis in three F2 populations, each containing a Rsp gene. These markers were converted into the sequence tagged site (STS) markers SUBC285, SOPC2, SOPAH5, and SOPBA12. Another STS marker (MWG938) linked to Rsp2 in coupling phase was also identified in an F2 population from the cross Robust/CIho 4780. The STS markers were tested on a set of 42 resistant and susceptible barley germplasm lines and 98 landraces. The expected sizes of marker fragments associated with each allele at Rsp loci were present in resistant or susceptible accessions. Efficiency of marker-assisted selection (MAS) for Rsp1, Rsp2, and Rsp3 using STS markers were evaluated in three F2--3 populations in the greenhouse and the field. Results of testing F2--3 progeny demonstrated that the accuracy of MAS was, with one exception, greater than 97% in the greenhouse and in two field locations (90% in the Osnabrock, ND trial for Rsp2). The STS markers closely linked to Rsp genes also identified the SSLB resistance corresponding to Rsp1, Rsp2, or Rsp3 in gene pyramiding F2 populations. The STS markers tightly linked to Rsp genes may be useful for M and for pyramiding with other genes in barley breeding for SSLB resistance.