This study was carried out to investigate and compare the levels of knowledge on Human African Trypanosomiasis (HAT), disease awareness among health personnel and factors affecting the diagnostic capacity of HAT in tsetse infested Chama and Mambwe districts of Eastern Zambia. Structured questionnaires were used to collect information from 110 health personnel drawn from 23 rural health centres (RHCs). Both districts reported low staffing levels for experienced laboratory personnel (7.7% for Chama and 12.2% for Mambwe). According to the survey, about 67.3% and 42.9% of the interviewed staff in Chama (n=52) and Mambwe (n=49), respectively, reported to carry out further investigations on patients that tested malaria negative (P =0.027). More staff from Chama district reported to have encountered HAT compared to Mambwe (P =0.000). About 88.5% (n=52) of the interviewed staff from Chama district were aware about the possible occurrences of HAT in their district (responses included abnormal sleep, headache, body pains, lymph node enlargement and microscopy) while the level of awareness in Mambwe district was 77.6% (n=49). The overall responses on the availability of basic laboratory tools for HAT diagnosis (including giemsa staining solution and microscopes) indicated that there was no significant difference (P>0.05) between the two districts although 43 suspected HAT cases were reported and 8 cases confirmed in Chama district between 2003 and 2013 while only one confirmed case was reported in Mambwe district during the same period. Both districts reported low collaboration with private sectors and low support from both government and private departments regarding the management of HAT compared to support received for malaria, HIV/AIDS and TB (P =0.007). In conclusion, health personnel from Chama and Mambwe districts had the potential to diagnose HAT but lacked sufficient support from both the government and private sectors to diagnose the disease.