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Background: Tomato yellow leaf curl virus (TYLCV) is a member of the genus Begomovirus in the family Geminiviridae, which causes severe losses in tomato production in tropic and subtropic regions., Methods: The purified TYLCV virions were used as the immunogen to produce monoclonal antibodies (MAbs) using the hybridoma technology. MAb-based dot enzyme-linked immunosorbent assay (dot-ELISA) and direct tissue blot immunoassay (DTBIA) were developed for sensitive, simple, and rapid detection of TYLCV in field tomato and whitefly (Bemisia tabaci) samples collected from TYLCV prevalent provinces in China., Results: Using the hybridoma technology, six murine MAbs (1C4, 8D10, 6E3, 2F2, 3F4 and 4G3) against TYLCV were prepared. Using the MAb 1C4, dot-ELISA and DTBIA were then established for detecting TYLCV in field tomato and whitefly samples collected from TYLCV prevalent provinces in China. The dot-ELISA could detect TYLCV in infected tissue crude extract diluted at 1:5,120 (w/v, g mL-1), and in viruliferous whitefly homogenate diluted at 1:128 (individual whitefly/μL), respectively. Field tomato samples (n=487) and whitefly samples (n=110) from TYLCV prevalent districts in China were screened for the presence of TYLCV using the two developed methods, and the results were further confirmed by PCR and nucleotide sequencing. The survey revealed that TYLCV is widespread on tomato plants in Zhejiang, Shandong and Henan provinces in China., Conclusions: The developed dot-ELISA is very suitable for the routine detection of TYLCV in field tomato and whitefly samples, and the DTBIA is more suitable for the routine detection of TYLCV in large-scale tomato plant samples collected from TYLCV prevalent areas.