The greater one-horned rhinoceros Rhinoceros unicornis is a flagship species for conservation in protected areas in India and Nepal. In India the species is afforded the highest level of legal protection under Schedule I of the Wildlife (Protection) Act 1972. Although censuses of greater one-horned rhinoceros have been carried out for decades using the traditional total count method, no advanced scientific approach has been adopted for population estimation of the species in India or elsewhere. We optimized noninvasive genetic techniques for identification of greater one-horned rhinoceros from dung samples, and applied these to estimate the number of rhinoceros in Gorumara National Park, in West Bengal, India. Our results confirmed the presence of 43 individuals from 60 dung samples collected throughout the Park in 2011. We confirmed a male-to-female sex ratio of 3.8 : 1, based on analysis of DNA from dung samples, using a y-chromosome linked marker. Our results are in concordance with a census carried out by the West Bengal Forest Department that found 42 rhinoceros in the Park, with a male-to-female sex ratio of 3.5 : 1. Our study thus demonstrates the feasibility of using a noninvasive genetic approach for population estimation of greater one-horned rhinoceros in the wild.