We investigated the feasibility and diagnostic utility of genotyping 9 CYP21 mutations, linked chromosome 6p markers, and a dimorphic X-Y marker from neonatal screening samples. Blood-impregnated filter papers (Guthrie cards) from 603 randomly chosen New Zealand neonates were genotyped blind to 17-hydroxyprogesterone (17-OHP) levels. Another 50 samples from Swiss and North American infants with correlative hormonal data were also genotyped. DNA was extracted, and gene-specific PCR was performed. CYP21 PCR products were subjected to ligase detection reaction, simultaneously analyzing 9 CYP21 mutations; PCR products of other genes were subjected to direct gel analysis. CYP21 genotyping indicated a heterozygote rate of 2.8% for classic mutations (excluding CYP21 deletions), and 2.0% for nonclassic mutations in New Zealanders. Ten full-term affected neonates showed a wide range of 17-OHP levels (15-1400 nmol/L). Sick or preterm infants or infants screened on the first day of life with high 17-OHP proved genetically unaffected. Genetic linkage disequilibrium was found between two CYP21 mutations and chromosome 6p markers. Guthrie cards can be used to accurately genotype CYP21 and other relevant markers, potentially enhancing the specificity and sensitivity of congenital adrenal hyperplasia screening. CYP21 heterozygote frequency for classic mutations is higher than expected based on genotype compared with that predicted by hormonal newborn screening.