Pathogenic variability of different isolates of Colletotrichum falcatum Went. collected from six varieties: Co 419, CoC 671 ( from Andhra Pradesh), Co 86032, Co87044, 89V74, and a local variety in Bhadrak (from Orissa) were established. Six red rot isolates were differentiated into four distinct pathotypes, viz., pathotype A - Cf 419 and Cf Bdkh; pathotype B - Cf 671; pathotype C - Cf 89V74 and pathotype D - Cf 86032, and Cf 87044. Pathotype A showed a susceptible reaction to Khakai, CoC 671, CoJ 64, and Co 419. Also pathotype B showed a susceptible reaction to Baragua, CoC 671, Co 997, CoJ 64, Co 1148, and Co 975, whereas pathotype C showed a susceptible reaction to CoC 671, Co 7717, Co 997, CoJ 64, Co 975, and Co 419. But pathotype D showed a susceptible reaction to CoC 671 and Co 997. Polymerase chain reaction (PCR) amplification and subsequent restricted fragment length polymorphism (RFLP) analysis of internal transcribed spacer region of ribosomal DNA (rDNA) of Cf 89V74, Cf 671 and Cf Vittal (C.falcatum isolate from Vittal, Karnatak) by four restriction enzymes, Alu I, Msp I, Rsa I, and Pvu II revealed two distinct groups viz.Group 1- Cf 89V74 and Cf Vittal; Group 2-Cf 671. Restriction enzymes Alu I, Msp I, Rsa 1, and Pvu II produced fragment lengths of 380, 520, 550, and 580 bp for Cf Vittal and Cf 89V74, whereas fragment lengths of 420, 370, 440, and 460 bp were observed in Cf 671, respectively. The present investigation with the red rot infected sugarcane samples clearly indicated the existence of two pathotypes viz., pathotype A and pathotype B in Andhra Pradesh and three pathotypes viz., pathotype A, pathotype C, and pathotype D in Orissa. Pathotype C (Cf 89V74) showed similar ITS- RFLP pattern in Cf Vittal which was isolated from Karnataka.