Red rot is one of the most wide spread sugarcane diseases in the country and has been a constraint on sugarcane productivity. Pathological as well as molecular studies were used to characterize the 11 isolates of Colletotrichum falcatum Went collected from sugarcane cultivars of different sugarcane-growing regions in northwestern states of India, to assess pathogen diversity. Seven reference pathotypes of C. falcatum from the northwestern zone of India were compared with four newly collected isolates of the same pathogen. All the newly collected isolates and existing pathotypes were inoculated on a set of 14 differentials in August 2011 by the plug method. After 60 days of inoculation, the observations were recorded and the pathotypes/isolates were categorized as resistant, intermediate, and susceptible according to the virulency behavior. On the basis of pathological categorization and comparison with reference pathotypes, it was concluded that the three isolates R1001 (CoJ 64), R1002 (CoS 88230), and R1004 (CoSe 92423) are similar to the existing isolate Cf 08, except for isolate R0401 from CoS 8436 (Shahjahanpur, Uttar Pradesh). This isolate differs from all the reference pathotypes of the northwestern zone of India indicating the existence of a new pathotype. Pathological results revealed that variety CoJ 64 is the ancestor/source of prevailing new races in nature because these three new isolates showed similarity with Cf 08, of CoJ 64. In this area, Cf 08 was widespread. The isolates were further tested for their variability with random amplification of polymorphic DNA (RAPD) primers. Twenty RAPD primers were screened, out of which seven gave amplification. Out of seven amplified primers, only two primers showed the polymorphism among 11 isolates (seven reference pathotypes and four new isolates) of C. falcatum. Analysis of the genetic coefficient matrix derived from the scores of RAPD profiles showed that minimum and maximum per cent similarities among the tested C. falcatum isolates existed in the range of 11.11 to 87.5, respectively. The dendogram analysis by the unweighted pair group method with arithmetic mean (UPGMA), separated two main clusters. The first cluster comprises only two isolates (Cf 07 & Cf 08), however the second cluster comprises all the other isolates (Cf 01, Cf 02, Cf 03, Cf 09, Cf 11, R1001, R1002, R1004 and R0401), confirming high genetic diversity among the isolates. The study also indicates the possibilities of a new isolate (R0401) in Shahjahanpur, which needs further investigation at the sequence level. The investigation is in progress.