Sugarcane bacilliform virus (SCBV), family Caulimoviridae, genus Badnavirus, is a virus harbouring a large genetic diversity and is usually asymptomatic on sugarcane commercial hybrids. While SCBV was one of the most prevalent viruses detected by High-Throughput Sequencing (HTS) at the Cirad quarantine, a new protocol for SCBV detection was established and applied. Primers amplifying a 579-bp fragment of the RT/RNase H viral region were designed based on SCBV sequences publicly available within the NCBI databases and SCBV sequences obtained from our HTS databases. The PCR protocol was setup and adapted to maximize SCBV detection using a large panel of sugarcane varieties and origins to detect SCBV isolates. Amplicons were Sanger sequenced and compared to known SCBV RT/RNase H partial sequences. This new protocol was successful for identifying SCBV isolates belonging to Badnavirus subgroups 1a and 1c (or clades 1 and 3), unique Badnavirus groups known to contain SCBV isolates so far. This novel broad spectrum SCBV detection protocol is now routinely used at the Cirad sugarcane quarantine, which made it an important tool for developing new cleaning processes against SCBV even if today only few countries consider SCBV as quarantine organism.