In summary, no software except MPP was capable of predicting primer sets for the larger target sets we examined or generating a multiplexed primer set without a MSA for any of the target sets we examined, even the smallest ones containing only a single species. Only HYDEN and GreeneSCPrimer, both requiring MSA inputs, completed for the two smallest target sets, selecting smaller sets of degenerate primers than the nondegenerate MPP primers. If target sets are sufficiently conserved so that a reasonable MSA can be built, and degenerate primers are acceptable, these tools may be preferable over MPP. However, for larger and more diverse target sets, only MPP completed.