Background: Spot blotch, caused by Cochliobolus sativus, is one of the most widespread and harmful diseases in barley. Identification of genetic loci associated with resistance to C. sativus is of importance for future marker-assisted selection. The goal of the current study was to identify loci conferring seedling resistance to two different pathotypes of C. sativus in the Siberian spring barley core collection. Results: A total of 96 spring barley cultivars and lines were phenotyped at the seedling stage with two C. sativus isolates (Kr2 and Ch3). According to the Fetch-Steffenson rating scale 16%/17% of genotypes were resistant and 26%/30% were moderate-resistant to the Kr2/Ch3 isolates respectively. A total of 94 genotypes were analyzed with the barley 50 K Illumina Infinium iSELECT assay. From 44,040 SNPs, 40,703 were scorable, from which 39,140 were polymorphic. 27,319 SNPs passed filtering threshold and were used for association mapping. Data analysis by GLM revealed 48 and 41 SNPs for Kr2 and Ch3 isolates, respectively. After application of 5% Bonferroni multiple test correction, only 3 and 27 SNPs were identified, respectively. A total of three genomic regions were associated with the resistance. The region on chromosome 3H associated with Ch3-resistance was expanded between markers SCRI_RS_97417 and JHI-Hv50k-2016-158003 and included 11 SNPs, from which JHI-Hv50k-2016-157070, JHI-Hv50k-2016-156842 had the lowest p-values. These two SNPs were also significant in case of Kr2 isolate. The region on chromosome 2H included 16 loci (7 of them with the lowest p-values were tightly linked to BOPA2_12_11504). Three loci corresponding to this region had suggestive p-values in case of Kr2 tests, so the locus on chromosome 2H may also contribute to resistance to Kr2 isolate. The third region with significant p-value in case of Kr2 tests was identified on chromosome 1H at the locus JHI-Hv50k-2016-33568. Conclusions: Three genomic regions associated with the resistance to one or both isolates of C. sativus were identified via screening of the Siberian spring barley core collection. Comparison of their location with QTLs revealed previously either with biparental mapping populations studies or with GWAS of distinct germplasm and other isolates, demonstrated that resistance to isolates Kr2 and Ch3 is conferred by known spot blotch resistance loci. Information on SNPs related can be used further for development of DNA-markers convenient for diagnostics of resistance-associated alleles in barley breeding programs.