Background - Recent advances in high-throughput genomics technology have expanded our ability to catalogue allelic variants in large sets of candidate genes related to premature coronary artery disease. Methods and Results - A total of 398 families were identified in 15 participating medical centers; they fulfilled the criteria of myocardial infarction, revascularization, or a significant coronary artery lesion diagnosed before 45 years in men or 50 years in women. A total of 62 vascular biology genes and 72 single-nucleotide polymorphisms were assessed. Previously undescribed variants in 3 related members of the thrombospondin protein family were prominent among a small set of single-nucleotide polymorphisms that showed a statistical association with premature coronary artery disease. A missense variant of thrombospondin 4 (A387P) showed the strongest association, with an adjusted odds ratio for myocardial infarction of 1.89 (P=0.002 adjusted for covariates) for individuals carrying the P allele. A variant in the 3′ untranslated region of thrombospondin-2 (change of thymidine to guanine) seemed to have a protective effect against myocardial in individuals homozygous for the variant (adjusted odds ratio of 0.31; P=0.0018). A missense variant in thrombospondin-1 (N700S) was associated with an adjusted odds ratio for coronary artery disease of 11.90 (P=0.041) in homozygous individuals, who also had the lowest level of thrombospondin-1 by plasma assay (P=0.0019). Conclusions - This large-scale genetic study has identified the potential of multiple novel variants in the thrombospondin gene family to be associated with familial premature myocardial infarction. Notwithstanding multiple caveats, thrombospondins specifically and high-throughput genomic technology in general deserve further study in familial ischemic heart disease.