AbstractApplications that require resequencing of hundreds or thousands of predefined genomic regions in numerous samples are common in studies of non-model organisms. However few approaches at the scale intermediate between multiplex PCR and sequence capture methods are available. Here we explored the utility of Molecular Inversion Probes (MIPs) for the medium-scale targeted resequencing in a non-model system. Markers targeting 112 bp of exonic sequence were designed from transcriptome of Lissotriton newts. We assessed performance of 248 MIP markers in a sample of 85 individuals. Among the 234 (94.4%) successfully amplified markers 80% had median coverage within one order of magnitude, indicating relatively uniform performance; coverage uniformity across individuals was also high. In the analysis of polymorphism and segregation within family, 77% of 248 tested MIPs were confirmed as single copy Mendelian markers. Genotyping concordance assessed using replicate samples exceeded 99%. MIP markers for targeted resequencing have a number of advantages: high specificity, high multiplexing level, low sample requirement, straightforward laboratory protocol, no need for preparation of genomic libraries and no ascertainment bias. We conclude that MIP markers provide an effective solution for resequencing targets of tens or hundreds of kb in any organism and in a large number of samples.