The sub-therapeutically use of tylosin by the swine industry provides selective pressure for the development of antibiotic resistance in gastrointestinal bacteria. Land application of swine manure into drained agricultural fields might accelerate the transport and dissemination of these microorganisms through soils, into tile drainage lines, and ultimately into surface waters; however, little is known about the hydrologic link between sources of antibiotic resistant bacteria and their transport in tile drainage systems. The objective of this study was to investigate the occurrence and transport of tylosin-resistant enterococci from tile-drained agricultural fields receiving semi-annual swine-waste applications. A field study was conducted at a farm near Nashua, IA in April (spring) and November (fall) of 2009. Liquid swine slurry from an operation feeding tylosin at sub-therapeutic levels was injected into no-till field plots. Resistance to tylosin in manure, soil and tile water was investigated by a phenotype-based method and polymerase chain reaction (PCR) using previously published primers, ermA, ermB, ermC, ermF, ermT, ermX, mefA, msrA . All enterococci in manure samples were resistant to tylosin in the spring, whereas about 68% of total enterococci in fall were resistant to tylosin. Average concentrations of total and tylosin-resistant enterococci in soil samples over the two experiments were 8.8 × 10⁶ cfu/g of soil and 8.9 × 10⁶ cfu/g of soil, respectively. Total and tylosin-resistant concentrations in tile water were significantly different during both the spring and fall experiments (P < 0.05). In drainage water, total enterococci ranged from 1.3 × 10¹ cfu/100 mL (fall) to 5.0 × 10³ cfu/100 mL (spring) while tylosin-resistant enterococci ranged from 1.3 × 10¹ cfu/100 mL (fall) to 1.2 × 10³ cfu/100 mL (spring). The greatest concentration of tylosin-resistant enterococci in manure and lowest in drainage water suggest that enterococci lose resistance as selective pressure from antibiotic residues decrease. Five macrolide-resistance genes namely ermB, ermC, ermF, ermT, msrAwere detected in greater than 9% of the samples. On average, most isolates (97%) harbored msrA, while only 9.5% of total isolates contains ermT gene and 9% contain ermC gene. The ermF gene was found in 156 out of 200 isolates (78%) and ermB gene was detected in 138 isolates (69%). Only four strains representing 2.0% of the total strains contains all five genes. The study results confirmed the hydrologic connection between the tylosin resistant enterococci and macrolide resistant genes in swine manure and their presence in tile water when manure is applied to agricultural fields followed by rainfall event. This work suggests that application of liquid manure from swine facilities administering sub-therapeutic doses of tylosin to tile-drained lands will promote the transport of antibiotic resistant bacteria through soil and tile lines, thus contaminating nearby soils and waters.