Cassava (Manihot esculenta) is a vital staple food crop in tropical regions, providing a significant source of nutrition and income for millions of people, especially in sub-Saharan Africa. However, Cassava brown streak disease (CBSD) poses a significant threat to its production, causing substantial yield losses and rendering cassava roots economically unusable. This study aims to validate the RT-PCR technique for the accurate and timely detection of Cassava brown streak virus (CBSV), the causative agent of CBSD. To achieve this, cassava leaf samples showing typical CBSD symptoms were collected from Mozambique, Tanzania, and Uganda. These samples were then subjected to RNA extraction using a modified Cetyl trimethylammonium bromide (CTAB) protocol at the Natural Resource Institute (NRI). The RNA was extracted and used as a template for reverse transcription-polymerase chain reaction (RT-PCR) with specific primers targeting CBSV. The results show successful amplification of CBSV partial coat protein sequences in all tested samples using CBSV 10 and 11 primers, confirming the presence of CBSV. Additionally, CBSV 9 and 11 primers successfully amplify full-length coat protein sequences from two samples, providing further validation of CBSV presence. These findings highlight the importance of RT-PCR as a sensitive and specific technique for detecting CBSV, providing a valuable tool for disease surveillance and management in cassava production. The study reinforces the significance of addressing CBSD to ensure food security and economic stability in regions that rely on cassava cultivation. Further research and implementation of RT-PCR-based diagnostic methods could help to reduce the socio-economic impact of CBSD and promote sustainable cassava production