Twinning is a complex trait with negative impacts on health and reproduction, which cause economic loss in dairy production. Several twinning rate quantitative trait loci (QTL) have been detected in previous studies, but confidence intervals for QTL location are broad and many QTL are unreplicated. To identify genomic regions or genes associated with twinning rate, QTL analysis based on linkage combined with linkage disequilibrium (LLD) and individual marker associations was conducted across the genome using high-throughput single nucleotide polymorphism (SNP) genotypes. A total of 9919 SNP markers were genotyped with 200 sires and sons in 19 half-sib North American Holstein dairy cattle families. After SNPs were genotyped, informative markers were selected for genome-wide association tests and QTL searches. Evidence for twinning rate QTL was found throughout the genome. Thirteen markers significantly associated with twinning rate were detected on chromosomes 2, 5 and 14 (P < 2.3 x 10(-5)). Twenty-six regions on fourteen chromosomes were identified by LLD analysis at P < 0.0007. Seven previously reported ovulation or twinning rate QTL were supported by results of single marker association or LLD analyses. Single marker association analysis and LLD mapping were complementary tools for the identification of putative QTL in this genome scan.