Sugarcane yellow leaf disease caused by Sugarcane yellow leaf virus (SCYLV) was spread over the world through infected germplasm. This study was conducted in order to determine the presence of SCYLV in commercial fields and germplasm collection and study the genetic diversity of the virus in Cuba. Leaf samples were collected from commercial cultivars in eight provinces and the Cuban germplasm collection in Matanzas province. The diagnosis of SCYLV was performed using tissue-blot immunoassay and reverse transcription polymerase chain reaction (RT-PCR). Our results revealed the widespread incidence of SCYLV in commercial sugarcane plantations in Cuba. The coat protein sequence analyses of SCYLV isolates under study confirmed the presence of BRA-PER, CUB and REU genotypes. The REU genotype is reported first time in Cuba. Coinfections of SCYLV genotypes (BRA-PER + CUB, BRA-PER + REU and CUB + REU) were also reported. The most predominating SCYLV genotypes reported in Cuba were identified as CUB and BRA-PER.