Prolonged severe vitamin D deficiency can cause osteomalacia, but the 25-hydroxyvitamin D (25OHD) concentration below which this occurs is unknown. We investigated the prevalence of biochemical osteomalacia in adults with a measurement of 25OHD. 25OHD results between 1/1/2009 and 15/6/2020 were obtained from the regional laboratory database, together with measurements of serum calcium, parathyroid hormone (PTH) and alkaline phosphatase (ALP) within 6months of the index 25OHD. We defined biochemical osteomalacia as all 3 of: albumin-adjusted serum calcium (aCa)<2.0mmol/L, PTH>7.3pmol/L and ALP>150IU/L. Possible osteomalacia was 2/3 criteria with the other test not done. 25OHD measurements associated with significant renal impairment, elevated hepatic transaminases or hypercalcaemia were excluded. 110,046 25OHD measurements were identified over the 11.5years period. After removal of ineligible measurements, 42,171 25OHD measurements from 32,386 individuals with at least 2 of aCa, PTH and ALP were included in analyses. Median 25OHD was 63nmol/L; 8% were <25nmol/L, and 33% were <50nmol/L. Five index 25OHD measurements met the definition of biochemical osteomalacia, and another 11 were possible osteomalacia. After reviewing available clinical records for these 16 episodes, we classified 9 cases as osteomalacia and 7 as other diagnoses. Thus, the prevalence of biochemical osteomalacia was 0.02% (9/42,171) for 25OHD measurements and 0.23% (8/3432) for 25OHD<25nmol/L. All cases of osteomalacia with 25OHD measurements prior to supplementation had 25OHD≤18nmol/L. The prevalence of biochemical osteomalacia is very low, even in individuals with 25OHD<25nmol/L.