Abstract The ability of an animal to develop a highly specific antibody response through affinity maturation has been considered an integral part of the adaptive immune response. However, much of the literature dealing with teleostean antibody responses suggests that little or no affinity maturation may occur within these taxa. As trout antibodies are similar to multimeric mammalian IgM, it has been reasoned that affinity maturational shifts in intrinsic affinity might be similarly small. Such small increases in affinity can, however, lead to potentially great avidity changes for multimeric antibodies. We therefore employed a partition-based immunoassay that permits the dissection of a single antiserum into discrete, affinity-based antibody subpopulations. Such partitioning assays provide for enhanced sensitivity and resolution of these affinity subpopulations over that which can be obtained by fluorescence quenching or equilibrium dialysis. Through the use of the partition-based immunoassay, we were able to detect a consistent increase in affinity within trout anti-TNP antisera. Furthermore, it was determined that trout are capable of generating new, higher affinity antibodies relatively late in the antibody response, which then come into dominance. Such evidence suggests that either somatic mutation does occur or that a unique form of affinity-based regulation of antibody expression is employed.