Riemerella anatipestifer is a gram-negative bacteria that can cause disease in a wide variety of wild and domesticated birds, especially waterfowl. The infection can be peracute, acute, or chronic. Although various routes of transmission have been proposed, to date, there is little information on the effects of route of transmission and challenge dosage on R. anatipestifer infection. Hence, the objective of this study was to determine the effect of route of inoculation and challenge dosage on R. anatipestifer infection and pathology. To achieve this objective, one hundred forty-seven 14-day-old white Pekin ducks (Anas platyrhynchos) were equally divided into 13 experimental groups (12 challenge and 1 control group). Each challenge group had 11 ducks. The control group had 15 ducks. Four routes of inoculation were evaluated (intranasal, oral, subcutaneous, and intravenous). Three dosage levels were evaluated for each inoculation route (10², 10⁴, and 10⁶ colony forming units [CFU]/ml). At the 10⁶ CFU/ml dosage level, mortality was most associated with the subcutaneous (91%) and intravenous (82%) routes, followed by the nasal (18%) and oral (9%) routes. A unique pathologic lesion was found in the bursa of Fabricius and spleen of affected birds. Within the spleen and bursa of Fabricius, there were varying degrees of lymphoid depletion and necrosis within the cortical and medullary regions. These pathologic lesions have not been previously reported in ducks with R. anatipestifer infection.