Background: Patellar luxation is an orthopedic disorder in which the patella moves out of its normal location within the femoral trochlea of the knee and it can lead to osteoarthritis, lameness, and pain. In dogs it is a heritable trait, with both environmental and genetic factors contributing to the phenotype. The prevalence of patellar luxation in the Dutch Flat-Coated Retriever population is 24%. In this study, we investigated the molecular genetics of the disorder in this population. Results: Genome-wide association analysis of 15,823 single nucleotide polymorphisms (SNPs) in 45 cases and 40 controls revealed that patellar luxation was significantly associated with a region on chromosome CFA07, and possibly with regions on CFA03, CFA31, and CFA36. The exons of the genes in these regions, 0.5 Mb combined, were analyzed further. These exons from 15 cases and a pooled sample from 15 controls were enriched using custom genomic hybridization arrays and analyzed by massive parallel DNA sequencing. In total 7257 variations were detected. Subsequently, a selection of 144 of these SNPs were genotyped in 95 Flat-Coated Retrievers. Nine SNPs, in eight genes on CFA07 and CFA31, were associated with patellar luxation (P <10(-4)). Genotyping of these SNPs in samples from a variety of breeds revealed that the disease-associated allele of one synonymous SNP in a pseudogene of FMO6 was unique to Flat-Coated Retrievers. Conclusion: Genome-wide association analysis followed by targeted DNA sequencing identified loci on chromosomes 7 and 31 as being involved in patellar luxation in the Flat-Coated Retriever breed.