p21(WAF1/CIP1), transcribed from the CDKN1A locus, plays a key role executing p53-induced growth arrest. The recent discovery that an alternative transcript, p21B, induces apoptosis, suggests an additional important function of this gene. Here, we report p21 and p21B mutation status in large cohorts of breast cancers and compare distributions of p21B polymorphisms in cancer patients to healthy controls. In 521 breast tumor samples analyzed, only one point mutation affecting the p21B protein was observed. No mutations were found when screening a panel of 20 established cell lines. A novel polymorphism, p21B(G128T) was identified. Haplotype analysis revealed no association between this variant and the previously identified p21B polymorphism p21B(T35C) or any of the known p21(WAF1/CIP1) polymorphisms. As previously reported for p21B(T35C), distribution of p21B(G128T) was similar among breast cancer patients and healthy controls (n = 691 and 1,015; incidence 6.1 vs. 4.8%; p = 0.273, respectively). No association between p21B(G128T) or p21B(T35C) and response to chemotherapy with an anthracycline-containing regimen or paclitaxel was recorded. Our findings do not suggest mutations or polymorphisms of p21B to play a major role with respect to either breast cancer risk or sensitivity towards chemotherapy.