Abstract: Background and Objective: Tomato Chlorosis Virus (ToCV) is a white fly-transmitted and phloem-limited crinivirus reported in this study for the first time in Egypt. ToCV caused drastic reduction in tomato yield since 2013. The aim of this study is to characterize the virus incidence using biological, serological and molecular tools. Materials and Methods: The B. tabaci MEAM1 white fly was used for virus isolation and propagation. Identity of ToCV , its natural hosts were confirmed with RT-PCR using a specific primer pair for ToCV-heat shock protein 70 homologue (HSP70h) gene, sequencing and phylogenetic studies. ToCV was purified using the innovative electro-elution technique. The induced antiserum for the Egyptian isolate of the virus (ToCV-Giza) was used for DAS-ELISA and dot blotting immuno-assays to evaluate the virus presence in tomato and other natural hosts. Results: The ToCV-Giza isolate was donated an accession number “MH667315.1” from the GenBank. Blastx sequence analysis of the HSP70h gene indicated 97-99% of amino acid similarities with many tested ToCV isolates. Phylogenetic studies showed the clustering of all ToCV isolates including ToCV-Giza in a separate group from the other tested criniviruses. The virus had a UV spectrum of a nucleoprotein with Amax and Amin at 260 and 240 nm, respectively and A260/280 ratio of 1.33. Out of 52 different tested plant species within 22 families, 44 were positive hosts for ToCV. Thirty seven out of these 44 plant species were considered as new hosts for ToCV in the present study. These included Ammi majus and Coriandrum sativum (Apiaceae), cabbage (Brassicaceae), sweet potato (Convolvulaceae), melon, cucumber, luffa (Cucurbitaceae), soybean, cowpea, faba bean (Fabaceae), Egyptian and American Cotton (Malvaceae). Several ornamentals either herbal type or woody trees belonging to Acanthaceae, Amaranthaceae, Euophorbiaceae, Moraceae and Rubiaceae were also recognized for the first time as hosts for ToCV. Conclusion: The obtained results confirmed the wide distribution of ToCV in its natural hosts in Egypt. Hygienic measures including control of the virus vector and removing of natural hosts should be strictly implicated.