The microbiota of the live production environment can directly shape the gastrointestinal microbiome of chickens and indirectly influence the health of birds. Therefore, numerous studies have attempted to characterize the microbial communities in litter and chicken feces from commercial poultry houses, but many treat a poultry house as a single sampling unit. Unfortunately, a poultry house has distinct areas (e.g., waterer/feeder, near fans, cooling pads), so effective sampling strategies need to be developed to account for this heterogeneity, especially when costlier microbiomic analyses are used to assess whole-house microbial diversity. Therefore, the goals of this study were to assess the spatial variability of the poultry litter and fecal microbiomes from distinct areas within a poultry house and to compare composite “whole-house” microbiomes pooled from all house areas, either (1) physically prior to DNA extraction or (2) combined in silico after sample processing (during DNA sequence analysis). No significant differences in α-diversity metrics (richness, diversity, evenness) were observed for fecal or litter microbiomes recovered from the different areas of the house, but β-diversity (litter only) and genus-level Operational Taxonomic Units (OTU) relative abundances (for fecal and litter) were found to be significantly different based on sampling area within the house. Additionally, the sample pooling method produced distinct composite fecal microbiomic profiles, but the litter microbiomes were unaffected. These results indicate that sample type, sampling area, and sample pooling method need to be carefully considered when determining appropriate sampling strategies for generating representative composite whole-house microbiomes for future microbiological-based live production studies.