The late diagnosis of HIV in patients across the UK is an increasing problem. Here, we report on a retrospective case-notes audit carried out to assess the impact of the 2008 UK HIV testing guidelines on clinical practice and identify missed opportunities for HIV testing. The audit was carried out in 2010 and focussed on patients with newly diagnosed HIV at centres providing adult HIV services across the UK. Data were collected on 1,112 patients, of whom 52.2% were found to have a late HIV diagnosis as defined as a CD4 T lymphocyte count of <350 cells/mm3. Most patients (62.6%) were diagnosed in traditional settings, with a significant increase in those diagnosed with HIV in non-traditional settings (33%) compared with the 2003 audit (18.5%) (p<0.001). The most frequent indicator conditions that patients had experienced were chronic diarrhoea or weight loss, sexually transmitted infection, blood dyscrasia or lymphadenopathy. A quarter of patients were identified as having had a missed opportunity for earlier diagnosis. Based on our results, we suggest that HIV testing needs to continue to expand across clinical settings to reduce the number of patients living with undiagnosed HIV infection.