The effective population size (N e ) is notoriously difficult to accurately estimate in wild populations as it is influenced by a number of parameters that are difficult to delineate in natural systems. The different methods that are used to estimate N e are affected variously by different processes at the population level, such as the life-history characteristics of the organism, gene flow, and population substructure, as well as by the frequency patterns of genetic markers used and the sampling design. Here, we compare N e estimates obtained by different genetic methods and from demographic data and elucidate how the estimates are affected by various factors in an exhaustively sampled and comprehensively described natural brown trout (Salmo trutta) system. In general, the methods yielded rather congruent estimates, and we ascribe that to the adequate genotyping and exhaustive sampling. Effects of violating the assumptions of the different methods were nevertheless apparent. In accordance with theoretical studies, skewed allele frequencies would underestimate temporal allele frequency changes and thereby upwardly bias N e if not accounted for. Overlapping generations and iteroparity would also upwardly bias N e when applied to temporal samples taken over short time spans. Gene flow from a genetically not very dissimilar source population decreases temporal allele frequency changes and thereby acts to increase estimates of N e . Our study reiterates the importance of adequate sampling, quantification of life-history parameters and gene flow, and incorporating these data into the N e estimation.T HE effective population size (N e ) is an essential concept in evolutionary and conservation biology as it determines the strength of stochastic evolutionary processes relative to deterministic forces (Crow and Kimura 1970). In the absence of gene flow, the rate of loss of genetic diversity via genetic drift is greater in populations with small N e . The effective population size is, however, notoriously difficult to accurately estimate in wild populations as it is influenced by a number of parameters that are difficult to characterize in natural systems. A number of different methods have been developed for estimating N e , and these are affected variously by different processes at the population level such as immigration, fluctuations in population size, population substructure, and life-history characteristics. When possible, it is therefore important to compare N e estimates obtained using different methods and elucidate how different processes affect the various methods for N e estimation (Fraser et al. 2007).Two main types of approaches have been used to estimate N e : genetic and demographic. Genetic methods attempt to infer the magnitude of the effective population size by characterizing the genetic consequences of limited population size, whereas demographic methods depend upon measurement of demographic parameters that have theoretically been shown to influence N e. The most common genetic approach is ...