OBJECTIVE To characterize the fecal microbiota of horses and to investigate alterations in that microbiota on the basis of sample collection site (rectum vs stall floor), sample location within the fecal ball (center vs surface), and duration of environmental exposure (collection time). ANIMALS 6 healthy adult mixed-breed mares. PROCEDURES From each horse, feces were collected from the rectum and placed on a straw-bedded stall floor. A fecal ball was selected for analysis immediately after removal from the rectum and at 0 (immediately), 2, 6, 12, and 24 hours after placement on the stall floor. Approximately 250 mg of feces was extracted from the surface and center of each fecal ball, and genomic DNA was extracted, purified, amplified for the V1-V2 hypervariable region of the 16S rDNA gene, and analyzed with a bioinformatics pipeline. RESULTS The fecal microbiota was unique for each horse. Bacterial community composition varied significantly between center and surface fecal samples but was not affected by collection time. Bacterial community composition varied rapidly for surface fecal samples. Individual bacterial taxa were significantly associated with both sample location and collection time but remained fairly stable for up to 6 hours for center fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that, for horses, fecal samples for microbiota analysis should be extracted from the center of fecal balls collected within 6 hours after defecation. Samples obtained up to 24 hours after defecation can be analyzed with the realization that some bacterial populations may deviate from those immediately after defecation.