We measured serum 25 hydroxyvitamin D [25(OH)D] levels of ambulatory adults in tropical Australia to determine whether it is appropriate to continue promoting sun-safety in this population. In August 2006 (winter), self-administered questionnaires were completed by 145 Meals-on-Wheels volunteers (49.3% male; mean age 57.8 ± 14.7 years; 76.6% response) from Townsville, Queensland (Latitude 19oS). Serum 25(OH)D was analyzed using two common assays. Mean levels were 68.3 (SD ± 18.7; range 26–142) by DiaSorin Radioimmunoassay and 83.0 (SD ± 30.8; range 30–184) by DiaSorin Liaison® one. No participants were 25(OH)D deficient (<25 nmol L−1). Nine participants (6.2%) had 25(OH)D levels between 25 and 50 nmol L−1 (insufficient), by both methods (seven with a BMI ≥ 25). Twenty-eight participants (19.3%) had one result in the insufficient range and the other in the adequate range. Thus, almost all of these free-living adults in tropical Australia had adequate vitamin D levels at the end of winter. There was poor agreement between the two 25(OH)D assays. These results suggest it is appropriate to continue promoting sun-safe messages to the ambulatory Caucasian adult population of North Queensland, which has an extremely high incidence of skin cancer. The lack of agreement between the two assays is a concern. Few doctors are aware of this measurement issue.