In Venezuela, cucurbit viruses have been associated with important yield losses. Therefore, an extensive survey was conducted to determine the major cucurbit viruses in this country. Leaf samples from 284 cucurbit plants exhibiting virus-like symptoms were collected mainly in 2009–2010 from several states of Venezuela. They were assessed for viral infection by polymerase chain reaction (PCR) for Melon chlorotic mosaic virus (MeCMV) and reverse transcriptase (RT)-PCR for Papaya ringspot virus (PRSV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucumber mosaic virus (CMV), Squash mosaic virus (SqMV) and Cucurbit aphid-borne yellows virus (CABYV). The most common virus in cucurbit fields, MeCMV, was present in 65·8% of samples. Its associated alphasatellite was found in 78% of samples positive for MeCMV. PRSV, ZYMV and WMV were found with different prevalence: 34·2, 32·4 and 1·1% respectively. CMV was also detected (6·7%) but SqMV and CABYV were not found. Single infections were more frequent than mixed infections (56·4 and 38·6%, respectively). For ZYMV, comparison and phylogenetic analyses of either polymerase and coat protein (NIb-CP) partial sequences or CP complete sequences revealed a low genetic diversity within Venezuelan isolates. Thirty-four ZYMV isolates were used for serological and biological analysis. Thirteen monoclonal antibodies showed a major group of isolates spread in several states and two groups located in Zulia only. Venezuelan ZYMV isolates showed biological variability on cucurbit cultivars susceptible, tolerant or resistant to ZYMV. Resistance to ZYMV in cucumber appears potentially durable, whereas resistance or tolerance in zucchini and melon may be easily overcome.