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The objective of this study was to determine the horizontal and vertical distribution of zooplankton in Harlan County Reservoir in order to assist in developing an appropriate zooplankton sampling regime for this and similar Nebraska irrigation reservoirs. Samples were collected at 16 sites distributed across 3 zones of the reservoir monthly in April, May, June, and July of 2007 using a 2.2 L Van Dorn sampler. Samples were collected at depths of 1, 4, and 7 meters, poured through an 80-μm filter, preserved, and identified to the lowest possible taxa. Total zooplankton densities in Harlan County Reservoir were statistically similar for samples collected at 1 m, 4 m, and 7 m of depth from April, June, and July samples. Samples collected in May had significantly more zooplankton at 1 m of depth (F = 6.98; p ≤ 0.01) compared to 4 m and 7 m. Density of total zooplankton collected at 1 m depth from pelagic and littoral sites in zone 1 was similar among months. Also, zooplankton densities were similar from the upper, middle, and lower regions of Harlan County Reservoir in all four sample months. These results indicate that zooplankton distribution in Harlan County Reservoir have a homogeneous vertical and horizontal distribution.