Tomato is one of the most important vegetable crops grown in many countries. With an increasing trend in greenhouse tomato production, growers are facing serious challenges from seed-borne and mechanically-transmitted viruses. A new tobamovirus, tomato brown rugose fruit virus (ToBRFV) first identified in 2014 in Jordan and Israel, has recently been reported on disease outbreaks of greenhouse tomatoes in southern California, USA and in Baja California, Mexico. Initial efforts to eradicate this emerging virus through total removal and hygiene practices have achieved some success. Unfortunately, new disease outbreaks were reported in other states. Several questions remain unanswered, including: 1) Was the virus introduced to North America through seeds or seedlings? 2) Were the outbreaks in Mexico and the U.S. resulted from the same or different introductions? In characterizing ToBRFV isolates in Mexico and the U.S., we identified two distinct types of genetic variants of ToBRFV. Understanding the genetic diversity of ToBRFV will improve the design of sequence-based detection tools. Due to cross serological reactions among tomato-infecting tobamoviruses, the potential presence of three additional tobamoviruses (i.e., tobacco mosaic virus, tomato mosaic virus and tomato mottle mosaic virus), and their varying levels of response to the resistance Tm2^2 gene, sensitive species-specific detection of ToBRFV is needed. In this study, we developed a RT-qPCR protocol, which will be useful for seed and plant health tests to assist in monitoring and controlling disease outbreaks.