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Meloidogyne floridensis is a recently described species of root-knot nematode (RKN) that is very closely related to many other tropical RKN species, including M. incognita. Despite its close phylogenetic relationship, M. floridensis is distinctive from its close relatives in both its meiotic mechanism of reproduction and its documented virulence on many of the most common RKN resistance genes in cultivated peach, tomato, and pepper. To further characterize the virulence profile of M. floridensis, we conducted replicate screens using this nematode to infect a panel of pepper lines that carry different sets of known RKN resistance genes. We found that M. floridensis was virulent against all the most common RKN resistance genes, including N, Me1, and Me3. We also found that two of these lines, PA 136 and PM 217, were highly resistant to M. floridensis. PA136 was previously considered to be universally susceptible to all other RKN species. Further testing of an F1 hybrid of this line confirmed this result and indicated that PA 136 contains a yet uncharacterized and potentially dominant source of species-specific resistance against M. floridensis. These surprising results provide additional data on the differences between M. floridensis and its close relatives, and identify new sources of resistance that could be used by pepper breeding programs to develop new cultivars with resistance against this nematode.