Abstract Background Twin and multiple births are rare in cattle and have a negative impact on the performance and health of cows and calves. Therefore, selection against multiple birth would be desirable in dairy cattle breeds such as Holstein. We applied different methods to decipher the genetic architecture of this trait using de-regressed breeding values for maternal multiple birth of ~ 2500 Holstein individuals to perform genome-wide association analyses using ~ 600 K imputed single nucleotide polymorphisms (SNPs). Results In the population studied, we found no significant genetic trend over time of the estimated breeding values for multiple birth, which indicates that this trait has not been selected for in the past. In addition to several suggestive non-significant quantitative trait loci (QTL) on different chromosomes, we identified a major QTL on chromosome 11 for maternal multiple birth that explains ~ 16% of the total genetic variance. Using a haplotype-based approach, this QTL was fine-mapped to a 70-kb window on chromosome 11 between 31.00 and 31.07 Mb that harbors two functional candidate genes (LHCGR and FSHR). Analysis of whole-genome sequence data by linkage-disequilibrium estimation revealed a regulatory variant in the 5ʹ-region of LHCGR as a possible candidate causal variant for the identified major QTL. Furthermore, the identified haplotype showed significant effects on stillbirth and days to first service. Conclusions QTL detection and subsequent identification of causal variants in livestock species remain challenging in spite of the availability of large-scale genotype and phenotype data. Here, we report for the first time a major QTL for multiple birth in Holstein cattle and provide evidence for a linked variant in the non-coding region of a functional candidate gene. This discovery, which is a first step towards the understanding of the genetic architecture of this polygenic trait, opens the path for future selection against this undesirable trait, and thus contributes to increased animal health and welfare.