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Estuaries are productive systems with rapid changes in natural stressors, such as salinity, that make perturbation detection challenging. Amphipods are vital to estuarine assemblages and may serve as anthropogenic stress indicators. However, practical methods of monitoring species composition and abundance are lacking. This study aims to describe the spatial patterns of four Gammarus spp. in northwest Atlantic estuaries and to compare their compositions and abundances between collection methods using artificial substrates, macrophyte raking, light-baited traps, and species-specific quantitative PCR analyses from sediment environmental DNA (eDNA). Sampling occurred in upper, mid, and lower estuary zones within three estuaries of Prince Edward Island (Canada). G. tigrinus was rarely found in the upper zones with any method. G. mucronatus was predominant in the upper–mid zones across the physical methods, and its abundance declined with increasing salinity. G. lawrencianus was a dominant species across zones, but its abundance did not change with salinity. G. oceanicus was predominant in lower-zone artificial substrates. Species abundances generally correlated with physical collection methods. Sediment eDNA did not detect the spatial effects observed via the physical methods but was correlated with the mean counts of G. mucronatus. The Gammarus spp. assemblages are spatially partitioned in short estuaries, though the sampling method is critical when interpreting estuary patterns. Though multiple methods are ideal for compositional comparisons, abundance monitoring should employ light traps. [ABSTRACT FROM AUTHOR]