Next-generation sequencing (NGS) technology and fast improvement in plant genetics have elevated to massive development of molecular genetic markers through fast analysis of huge molecular biological data. Furthermore, in domestic commercial breeding of horticultural crops, the application of marker assisted breeding (MAB) has been introduced recently. For effective improvement of cultivar breeding, in this research, transcriptome analysis and single nucleotide polymorphism (SNP) comparison with high density pepper map in UC-DAVIS were performed using four lines of Capsicum annuum and C. chinense. For rapid analysis of MAB of tolerant pepper lines, 412 Fluidigm probes were newly designed in this study. These designed probes and SSR and COSII markers were applied for background selection through the MAB program. In addition, powdery mildew (PM), tomato spot wilt virus (TSWV) resistance related markers were subjected to foreground selection of BC1, BC2, and BC3 progenies. The MAB system using Fluidigm probes, and trait-related and common markers was introduced into domestic pepper breeding, which will rapidly approach to a new elite line and a commercial tolerant cultivar.