ABSTRACT: Acoustic echosounders collect detailed information on the location of patchily distributed pelagic organisms over varying spatial scales. This study measured the spatial variability of epi- and mesopelagic 38 kHz backscatter along the US Mid- and South Atlantic continental shelf and slope. We used variogram analysis to estimate the horizontal spatial structure of backscatter measurements, examined whether environmental variables might affect these estimates, and assessed potential impacts of acoustic survey design. Backscatter data were collected during ship-based surveys (50-100 km²) at 7 sites during 4 cruises between November 2017 and November 2019. Average patch size estimates were consistently between 2 and 4 km among locations. Modeled variogram range varied significantly with the depth of the backscatter layer, but linear effect sizes were negligible (<1 m). Chlorophyll a (chl a) concentration had a significant positive effect on range (95 m), suggesting that patch sizes are slightly larger in the epipelagic where chl a concentration is higher. Incorporating variogram parameters of range, sill, and nugget produced some clustering of spatial correlation parameters with scattering layer depth, particularly for the deepest sites assessed (700-900 m depth). Spatial characteristics of a given location were not significantly different between surveys of the same size, but sometimes differed with smaller (25% of area) survey sizes. These results offer insight into nekton and macrozooplankton backscatter patterns in important shelf break and slope systems across horizontal and vertical dimensions, and provide needed information for monitoring fine- to mesoscale offshore marine habitat areas.