Objective: To determine, through the use of molecular diagnostic tools, whether the two species of parasite that cause human African trypanosomiasis have become sympatric. Design: Blood sampling of all available patients between June 2001 and June 2005 in central Uganda and between July and September 2003 in northwest Uganda and analysis of subcounty sleeping sickness records in Uganda between 1985 and 2005. Setting: Sleeping sickness treatment centres in central and northwest Uganda and in south Sudan. Participants: Patients presenting at the treatment centres and diagnosed as having sleeping sickness. Main outcome measure: Classification of parasites from patients from each disease focus as either Trypanosoma brucei rhodesiense (acute form) or T b gambiense (chronic form). Results: Blood from 231 patients with sleeping sickness in central Uganda and from 91 patients with sleeping sickness in northwest Uganda and south Sudan were screened for T b rhodesiense (detection of SRA gene) and T b gambiense (detection of TgsGP gene). All samples from central Uganda were classified as T b rhodesiense, and all samples from northwest Uganda and south Sudan were identified as T b gambiense. Conclusions: The two focuses of human African trypanosomiasis remain discrete, but the area of Uganda affected by the acute form of human sleeping sickness has increased 2.5-fold since 1985, spreading to three new districts within the past five years through movement of infected livestock. Without preventive action targeted at the livestock reservoir of this zoonotic disease, it is likely that the two disease focuses will converge. This will have a major impact on diagnosis and treatment of this neglected disease. Real time monitoring is recommended, using molecular diagnostic tools (at a regional surveillance centre, for example) targeted at both livestock and human patients.