In this study, non-invasive molecular methods were used to investigate the abundance and spatial organisation of otters (Lutra lutra) in Kinmen. DNA samples were extracted from fresh spraints collected seasonally along two streams from February to November 2001 and genotyped using a panel of 7 microsatellites and the SRY gene. Out of 343 spraints, 222 were successfully genotyped and 38 different genotypes (19 females and 19 males) were identified. Thirteen of these were residents that were identified in more than one season and 25 were floaters that were only identified in a single season. The average number of otters per km found along the two streams (1.5–1.8 for all otters, or 0.8–1.1 for residents only) was higher than that estimated in other studies. Female residents lived in exclusive group ranges and the ranges of male residents overlapped with no more than one group range of female residents. Otters appearing in the same range tended to be more closely related to each other. This study demonstrates that non-invasive molecular methods can be used to reveal a more comprehensive estimation of size and structure in an otter population.