Septoria speckled leaf blotch (SSLB), caused by Septoria passerinii, has become one of the most serious diseases of barley in the Upper Midwest region of the USA. The recombinant line 36L5 derived from a backcross of the susceptible barley cultivar 'Emir' and a resistant Hordeum bulbosum parent Cb2920/4/Colch was found to be resistant to S. passerinii. Two doubled haploids derived from 36L5 were backcrossed to cv. 'Emir' to obtain two BCF2 populations for determining the inheritance of resistance to S. passerinii. BCF2 progeny and BCF2: 3 families were evaluated at the seedling stage in the greenhouse for reaction to S. passerinii. BCF2 progeny and BCF2: 3 families from both crosses segregated 3 : 1 (resistant : susceptible), and 1 : 2 : 1 (resistant : segregating : susceptible), respectively, indicating that the H. bulbosum-derived SSLB resistance is conferred by a single dominant gene. The H. bulbosum introgressions were positioned on chromosome 4HL by genomic and fluorescent in situ hybridizations (GISH and FISH, respectively) and by Southern hybridization with the rye repetitive sequence pSc119.2. These findings indicate that SSLB resistance in H. bulbosum has the potential to be transferred and utilized in barley breeding programs.