The root-knot nematode problem in guava is a newly emerging problem in India. A new report of the presence of new invasive species of root-knot nematode Meloidogyne enterolobii is of major concern now. Representative samples collected from five major guava growing districts namely, Bengaluru, Chikkaballapur, Dharwad, Kolar, and Shivamogga of Karnataka state, India were used for molecular characterization using ITS primer and M. enterolobii specific SCAR primer. All the samples confirmed the presence of new invasive species of root-knot nematode (M. enterolobii) in guava orchards located in different parts of Karnataka. Phylogenetic analysis using the maximum likely hood method grouped the eight isolates into two sub-clusters under a single clade. To the best of our knowledge, the present investigation is the first molecular based confirmation of the presence of M. enterolobii in Karnataka.