A growing number of equine studies examine management factors that influence the microbial populations in regions of the gastrointestinal tract using culture-independent molecular techniques. We used quantitative polymerase chain reaction (qPCR) to evaluate changes in fecal streptococcal populations in horses fed different maturities of teff hay with fecal samples collected at 7 AM, 12 PM, and 7 PM. The objective of this study was to use qPCR and species-specific probes for 16S ribosomal DNA to quantify the percentage of equine hindgut streptococcal species (EHSS) relative to the total bacterial load in the feces. Feces from horses fed the most mature teff hay had the lowest %EHSS, and feces collected at 12:00 had the highest %EHSS (P < .05). Our interest in investigating %EHSS developed out of research that examined associations between changes in EHSS populations and the onset of laminitis. Although we expected almost no risk of laminitis in these forage fed horses, our hypothesis was that the different carbohydrate fractions in the three maturities of hay would result in differences in %EHSS in horses fed these hay maturities. The supply of carbohydrates, nonstructural or structural, influences the microbial species composition through the gastrointestinal tract. This study highlights the occurrence of measurable changes in %EHSS due to subtle changes in dietary nonstructural carbohydrate and also changes in samples taken at different times of day. This information is useful to others considering investigations of dietary influences on gastrointestinal microbial populations, particularly those that plan to use feces as their main sample medium.