Altogether 71 samples collected in 1968–2004 from the Rokytná and Loučka rivers were used to estimate proportional fish species richness from single-pass electro-fishing and probabilities of detection for individual fish species. Mean estimated species richness from single-pass sampling (p ́s1) was 95.4% and 96.2% of estimated total species richness for Rokytná and Loučka rivers, respectively. However, p ́s1 values for individual sites ranged from 71.4% to 100% of estimated total species richness. Additional species unique to the second pass were collected in 38% (Rokytná) and 22% (Loučka) of the samples. When assessing the fish assemblage structure by a multivariate technique (PCA), considerable inaccuracy between the single-pass and two-pass electro-fishing data was also observed. Decisions regarding standardized sampling effort and whether to conduct one or more electro-fishing passes must be based on study objectives. For more accurate evaluations of fish assemblage structure two-pass electro-fishing is recommended. Three-pass electro-fishing did not produce significant increase of species richness.