The MUTYH gene is involved in base excision repair. MUTYH mutations predispose to recessively inherited colorectal polyposis and cancer. Here, we evaluate an association with breast cancer (BC), following up our previous finding of an elevated BC frequency among Dutch bi-allelic MUTYH mutation carriers. A case–control study was performed comparing 1,469 incident BC patients (ORIGO cohort), 471 individuals displaying features suggesting a genetic predisposition for BC, but without a detectable BRCA1 or BRCA2 mutation (BRCAx cohort), and 1,666 controls. First, for 303 consecutive patients diagnosed before age 55 years and/or with multiple primary breast tumors, the MUTYH coding region and flanking introns were sequenced. The remaining subjects were genotyped for five coding variants, p.Tyr179Cys, p.Arg309Cys, p.Gly396Asp, p.Pro405Leu, and p.Ser515Phe, and four tagging SNPs, c.37-2487G>T, p.Val22Met, c.504+35G>A, and p.Gln338His. No bi-allelic pathogenic MUTYH mutations were identified. The pathogenic variant p.Gly396Asp and the variant of uncertain significance p.Arg309Cys occurred twice as frequently in BRCAx subjects as compared to incident BC patients and controls (p=0.13 and p=0.15, respectively). The likely benign variant p.Val22Met occurred less frequently in patients from the incident BC (p=0.03) and BRCAx groups (p=0.11), respectively, as compared to the controls. Minor allele genotypes of several MUTYH variants showed trends towards association with lobular BC histology. This extensive case–control study could not confirm previously reported associations of MUTYH variants with BC, although it was too small to exclude subtle effects on BC susceptibility.