Turner syndrome (TS) occurs when an X-chromosome is completely or partially deleted, or when X-chromosomal mosaicism is present. Girls with TS benefit from early diagnosis and treatment with growth hormone; however, many girls with TS are not detected until after 10 years of age resulting in delayed evaluation and treatment. To facilitate the detection of TS, we developed a high-throughput test for TS, based on a quantitative method of genotyping to detect X-chromosome abnormalities. This test utilizes pyrosequencing to quantitate relative allele strength (RAS) from single nucleotide polymorphisms (SNPs) using 18 informative SNP markers that span the X-chromosome and one marker for the detection of Y-chromosome material. To determine the validity of this rapid test for TS detection, we undertook a large-scale study using DNA from 132 females without TS and 74 females with TS for whom karyotypes were available. TS was identified with 96.0% sensitivity and 97.0% specificity in this cohort. We also tested buccal swab DNA from a group of 72 females without TS and 69 females with TS. In this group, TS was identified with 97.1 sensitivity and 90.3% specificity. These results demonstrate the validity of a high-throughput, pyrosequencing based test for the accurate detection of TS, providing a potential alternative to karyotype testing.