ABSTRACT Rapid and cost-effective genotyping of large mapping populations can be achieved by sequencing a reduced representation of the genome of every individual in a given population and using that information to generate genetic markers. A customized genotyping-by-sequencing (GBS) pipeline was developed to genotype a rice F2 population from a cross of Oryza sativa ssp. japonica cv. Nipponbare and the African wild rice species Oryza longistaminata . While most GBS pipelines aim to analyze mainly homozygous populations we attempted to genotype a highly heterozygous F2 population. We show how species-and population-specific improvements of established protocols can drastically increase sample throughput and genotype quality. Using as few as 50,000 reads for some individuals (134,000 reads on average) we were able to generate up to 8,154 informative SNP markers in 1,081 F2 individuals. Additionally, the effects of enzyme choice, read coverage and data post-processing are evaluated. Using GBS-derived markers we were able to assemble a genetic map of 1,536 cM. To demonstrate the usefulness of our GBS pipeline we determined QTL for the number of tillers. We were able to map four QTLs to chromosomes 1, 3, 4 and 8 and confirm their effects using introgression lines. We provide an example of how to successfully use GBS with heterozygous F2 populations. By using the comparatively low-cost MiSeq platform we show that the GBS method is flexible and cost-effective even for smaller laboratories