In a previous study, the 30-year effect (1970 to 2000) of a 25,000-head beef cattle feedyard upon a downwind native shortgrass pasture was documented. Vegetation changes and a soil phosphorus (P) gradient with respect to distance from the feedyard were determined. The objective of this study was to evaluate the effects of vegetation, soil depth, and soil P on soil microbial populations using denaturing gradient gel electrophoresis-polymerase chain reaction (DGGE-PCR) employing universal prokaryotic 16S PCR primers 1092F and 1392R. Vegetation (% ground cover and plant type) and soil P levels were determined at 3 soil depths (0-2, A; 4-6, C; and 15-30 cm, E) at 119 locations (22.5 by 60 m grid). Six soil P classes (A – F based on soil P values for each depth) and three vegetation classes (based on % of plant types, AF – annual forbs, PG – perennial grasses, and MX – mixed % of AF and PG) were determined for the categorization of locations distal to the feedyard. Community DNA samples were prepared from each composite sample (eight cores pulled and composited per location) using soil DNA extraction kits. The effect of vegetation class, soil P class, and soil depth on microbial community structure data reveal that soil depth and plant vegetation patterns were observed to influence microbial populations. The most homogeneous microbial populations were observed at soil depth E (32.8% similarity), followed by C (14.2%), and A (10.1%). Overall homogeneity of the three soil depths was 13.4% similarity. The effect of vegetation was most pronounced on the C soil depth with little apparent effect on soil depth A. PG were associated with two main DNA groups with an overall similarity of 34.2% with several subgroups that diverged at 62.4, 70.1, and 70.2 % similarity. AF was associated with one microbial group with a similarity of 67.3%.