The World Health Organization targeted () human African trypanosomiasis for elimination of transmission by 2030. Sensitive molecular markers that specifically detect type 1 () parasites will be important tools to assist in reaching this goal. We aim at improving molecular diagnosis of 1 infections by targeting the abundant mitochondrial minicircles within the kinetoplast of these parasites. Using Next-Generation Sequencing of total cellular DNA extracts, we assembled and annotated the kinetoplast genome and investigated minicircle sequence diversity in 38 animal- and human-infective trypanosome strains. Computational analyses recognized a total of 241 Minicircle Sequence Classes as 1-specific, of which three were shared by the 18 studied 1 strains. We developed a minicircle-based assay that is applicable on animals and as specific as the -based assay, the current golden standard for molecular detection of 1. The median copy number of the targeted minicircle was equal to eight, suggesting our minicircle-based assay may be used for the sensitive detection of 1 parasites. Annotation of the targeted minicircle sequence indicated that it encodes genes essential for the survival of the parasite and will thus likely be preserved in natural 1 populations, the latter ensuring the reliability of our novel diagnostic assay.