Non‐invasive genetic analysis has been frequently employed to estimate ecological and population parameters for many secretive and/or threatened species. However, Neotropical carnivores have so far been scarcely targeted by such studies. The Neotropical otter (Lontra longicaudis) is a poorly‐known species for which local levels of genetic diversity and demographic parameters are virtually absent. We employed non‐invasive sampling and amplification of microsatellite loci to investigate population size and density, spatial organization, and relatedness of a wild Neotropical otter population in an Atlantic forest area in southern Brazil. We directly identified 28 individuals and estimate a rather high population density at the study site. Spatial organization analysis indicated that male cumulative displacement was higher than that of females, with the latter sex showing evidence of philopatric behaviour. Also, the reconstruction of genealogical relationships suggests that spatial organization in this otter appears to be influenced by relatedness. By allowing the testing of specific hypothesis targeting these issues, our results provided important glimpses into the Neotropical otter's population biology. Moreover, the findings of the present study reaffirm the power of non‐invasive genetics to investigate the biology of this elusive species, and open up new avenues for ecological and demographic studies of other Neotropical carnivores. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109, 932–948.