Solanum lycopersicoides is a wild nightshade relative of tomato with known resistance to awide range of pests and pathogens, as well as tolerance to cold, drought and salt stress. Toeffectively utilize S. lycopersicoides as a genetic resource in breeding for tomato improvement,the underlying basis of observable traits in the species needs to be understood. Molecularmarkers are important tools that can unlock the genetic underpinnings of phenotypicvariation in wild crop relatives. Unfortunately, DNA markers that are specific to S. lycopersicoidesare limited in number, distribution and polymorphism rate. In this study, we developeda suite of S. lycopersicoides-specific SSR and indel markers by sequencing, building andanalyzing a draft assembly of the wild nightshade genome. Mapping of a total of 1.45 Gb ofS. lycopersicoides contigs against the tomato reference genome assembled a moderatenumber of contiguous reads into longer scaffolds. Interrogation of the obtained draft yieldedSSR information for more than 55,000 loci in S. lycopersicoides for which more than 35,000primers pairs were designed. Additionally, indel markers were developed based on sequencealignments between S. lycopersicoides and tomato. Synthesis and experimental validation of345 primer sets resulted in the amplification of single and multilocus targets in S. lycopersicoidesand polymorphic loci between S. lycopersicoides and tomato. Cross-species amplificationof the 345 markers in tomato, eggplant, silverleaf nightshade and pepper resulted invarying degrees of transferability that ranged from 55 to 83%. The markers reported in thisstudy significantly expands the genetic marker resource for S. lycopersicoides, as well as forrelated Solanum spp. for applications in genetics and breeding studies.