Preserving genetic diversity and preventing its loss in livestock populations is essential for further sustainable development and breeding. The main purposes of this study were to estimate the current status of genetic diversity of the approved genetic resources (GRs) in indigenous Czech Red (CR) and Czech Spotted (CS) cattle and assess the impact of diversifying selection on two CR subpopulations based on genome-wide single-nucleotide polymorphism (SNP) analysis. The main within-breed genetic diversity indicators of CR and CS (share of polymorphic SNPs, observed heterozygosity, and minor allele frequency) are intermediate or high, and the inbreeding coefficients are below zero. The small degree of genetic differentiation (maximum FST = 0.088) suggests a low divergence between the breeds and populations. Principal component analysis (PCA) shows a less genetic variation in CS and, in contrast, larger variability within and genetic similarity between the two analysed CR subpopulations. Putative selection signatures based on FST revealed that the ten most differentiated genomic regions between the two CR subpopulations were located on chromosomes 1, 3, 11, 12, 18, and 22, encompassing only 7 Mb of sequence and 69 protein-coding genes. Selection differences are low and probably attributed to the influence of the CS genome exploited in CR cattle revitalization. The majority of the candidate genes are involved in the immune system response, fertility, and neuronal and brain function. Gene Ontology enrichment and quantitative trait loci database analyses show that the genes act in milk and meat production and reproductive performance. These findings expand our knowledge about the genomic diversity of endangered Czech indigenous cattle and provide helpful information to organize their breeding programmes and preservation.