The objective of this longitudinal controlled trial was to quantitatively compare carriage of a gene encoding for ceftiofur-resistance (bla(CMY-2)), standardized to a reference gene (16SrRNA), among total community DNA extracted from fecal samples collected from cattle treated with three different dose regimens of ceftiofur crystalline-free acid (CCFA) versus those untreated (controls). Sixty-one steers were assigned to three treatment regimens and housed in six pens. In each pen, five steers were treated and five were controls (one of the pens had six controls). CCFA administration was as follows: two-thirds dose treatment (4.4 mg/kg, on day 0), single-dose treatment (6.6 mg/kg, on day 0), and three-dose treatment (6.6 mg/kg, on days 0, 6, and 13). Fecal samples were collected on days 0, 3, 7, 10, 14, 18, 21, and 28. The gene copy numbers/gram of feces for bla(CMY-2) and 16SrRNA were determined in total community DNA samples using quantitative real-time PCR. The relationships between the quantities of standardized bla(CMY-2), nonstandardized bla(CMY-2), and nonstandardized 16SrRNA, and the explanatory variables (treatment, time, and treatment x time) were assessed using repeated measures mixed models. There were significant differences in each of the three models with respect to each explanatory variable. Overall, while steers administered three doses and two-thirds dose of CCFA had significantly higher quantities of nonstandardized bla(CMY-2) than controls, the standardized values were lower. The administration of CCFA in feedlot cattle may provide selection pressure favoring higher levels of bla(CMY-2) carriage, but this may also lead to concurrent reductions in the total bacterial population (as reflected by lowered 16SrRNA) during the treatment period.