Routine clinical evaluation of β-globin phenotypes usually includes HPLC screening and alkaline/acid electrophoresis and/or isoelectric focusing. Although common structural variants are easily identified in the routine environment, specific β-thalassemia mutations are not and these can be the basis for prognosis and also for subsequent prenatal diagnosis for at-risk family members. Herein, we report evaluation of the performance of a linear array-based β- hemoglobinopathy mutation assay provided by Roche Molecular Diagnostics (Pleasanton, CA) that detects 6 β-globin variants (S, C, E, Knossos, D-Los Angeles and O-Arab) and 42 β-thalassemia mutations (90% predicted detection in any at-risk population group). The assay consists of one multiplex PCR with biotinylated primers, amplicon hybridization to a reverse line blot array, and colorimetric detection of a streptavidin-HRP conjugate. We tested 50 archived, abnormal and 92 sequential clinical samples, all previously tested in our routine assays. The results of the DNA and HPLC/electrophoresis were 100% consistent with the linear array, although Hb-S, C, D-Los Angeles and E were the only structural variants present in our collection. We verified the presence of Hb-S and excluded Hb-O-Arab in an ambiguous compound heterozygote for which HPLC and electrophoresis could not distinguish possible Hb-C-Harlem from Hb-O-Arab. Of 15 putative β-thalassemia minor samples, 10 had 6 different β-thalassemia mutations. Based on our experience, the daily throughput for a single technologist performing this mutation analysis is approximately 200 samples; it is, thus, an appropriate second-tier assay for newborn screening programs. The assay is robust, reliable and is also an excellent screen prior to sequencing for rare mutations.