Antibodies provide some protection against cytomegalovirus-mediated disease. All aspects of antibody recognition of important viral antigens are, however, not fully appreciated. Glycoprotein B (gB), a key protein in the viral membrane, participates in viral infection and it is a component of prototype vaccines. By using combinatorial antibody library and selection technology, novel antibody specificities targeting gB have now been isolated. We define a monoclonal antibody fragment able to recognize site I of antigenic domain (AD) 2, a poorly immunogenic epitope targeted by potent virus-neutralizing antibodies, in a way that is different from the binding of antibodies induced by infection but similar to those induced by vaccination. We also describe the existence of a novel epitope overlapping site I of AD-2 and AD-1, the immunodominant epitope of gB. These specificities, derived by molecular engineering, will be useful for the future assessment of humoral immune responses against this opportunistic viral infection.