Watermelon (Citrullus lanatus) and other cucurbits are important crops grown in Guatemala for local consumption and export. The whitefly (Bemisia tabaci) vector of Cucurbit yellow stunting disorder virus (CYSDV), Melon chlorotic leaf curl virus (MCLCuV), and Squash vein yellowing virus (SqVYV) was observed in fields with numbers increasing during the season. Four samplings of crowns, peduncles, and/or leaves of symptomatic plants were made in March and April 2015. Total RNA was extracted from symptomatic plant tissue and tested by RT-PCR for SqVYV, CYSDV, Papaya ringspot virus (PRSV), and/or begomoviruses. Primers specific for the coat protein gene of SqVYV (1020 bp), CYSDV (707 bp), or PRSV (511 bp), and degenerate begomovirus primers (1159 or 533 bp) amplified products of the expected sizes from 15 of 24, 20 of 24, 4 of 24, or 8 of 8 plants, respectively. SqVYV amplicons from six individual plants from the fourth sampling, SqVYV and CYSDV amplicons from a pool of plants from the third sampling, and degenerate begomovirus amplicons from the second sampling were cloned in the pGEM-T vector. Five clones of each amplicon were sequenced in both directions and representative consensus sequences were deposited in GenBank (Accession Nos. KT007178 to KT007183). Sequence analysis demonstrated that SqVYV coat protein gene sequences from Guatemala shared 99 to 100% nucleotide (nt) identity with each other, and 97 to 98% nt identity with divergent SqVYV isolates previously described from Florida (e.g., WM2005aHi, GenBank Accession No. JF897974) and California (GenBank Accession No. KP218061), but only 90% nt identity with the predominant SqVYV isolate found in Florida (e.g., Sq2003Hi, GenBank Accession No. EU259611) . Tissue blots were prepared from crowns and peduncles from the first, third, and fourth samplings, and tested by tissue blot nucleic acid hybridization assay for SqVYV. Tissue blots indicated SqVYV infection in an additional 48 of 102 watermelon samples, and cylindrical inclusions typical of SqVYV were observed in phloem tissue from the fourth sampling by light microscopy, confirming the identification of SqVYV. This is the first report of SqVYV infecting watermelon in Central America. Resources in this dataset:Resource Title: First Report of Squash vein yellowing virusin Watermelon in Guatemala. File Name: Web Page, url: https://apsjournals.apsnet.org/doi/epdf/10.1094/PHP-BR-15-0019