Two major quantitative trait loci (QTLs) associated with resistance to powdery mildew (Blumeria graminis f. sp. hordei) were previously identified on chromosome 7H of the Spanish barley line SBCC097. The two QTLs seemed to share the same chromosomal position as the major genes mlt and Mlf, which were formerly described in Hordeum vulgare ssp. spontaneum-derived lines. In the present work, different lines that carry mlt (RS42-6*O), Mlf (RS137-28*E), or a combination of both (SI-4 and SI-6) were compared with SBCC097 to evaluate their relatedness at the phenotypic, cellular, and genetic levels. The resistance of the lines was characterised by inoculating them with a set of 27 isolates of B. graminis, which displayed a wide range of virulence. It was revealed that SBCC097 possessed a distinctive resistance spectrum. Microscopic assessment of the cytological development of the resistance response showed that SBCC097 clearly formed fewer well-established colonies and secondary hyphae than the other lines. This was confirmed by the infection type recorded after visual inspection. Genetic analyses of all five lines, based on markers flanking the QTLs derived from SBCC097, supported the macroscopic and microscopic data and pointed to the presence of a combination of novel genes or alleles in SBCC097, which may be included in the category of “intermediate-acting” genes, governing resistance mainly at the post-penetration stage.