Five new phlebotomus fever virus serotypes (Bunyaviridae: Phlebovirus) are described. These viruses, designated Ambe, Ixcanal, Mariquita, Armero, and Durania, were isolated from sand flies (Diptera: Psychodidae) collected in Brazil, Colombia, and Guatemala. Two of the agents were recovered from pools of male sand flies. The new viruses are antigenically related to other members of the phlebotomus fever serogroup by immunofluorescence, but are distinct from the other 39 members of this serogroup by plaque reduction neutralization test.