The Lotus japonicus symbiont Mesorhizobium loti R7A encodes two copies of nodD and here we identify striking differences in Nod factor biosynthesis gene induction by NodD1 and NodD2 both in vitro and in planta. We demonstrate that induction of Nod factor biosynthesis genes is preferentially controlled by NodD1 and NodD2 at specific stages of symbiotic infection. NodD2 is primarily responsible for induction in the rhizosphere and within nodules, while NodD1 is primarily responsible for induction within root hair infection threads. nodD1 and nodD2 mutants showed significant symbiotic phenotypes and competition studies establish that nodD1 and nodD2 mutants were severely outcompeted by wild-type R7A, indicating that both proteins are required for proficient symbiotic infection. These results suggest preferential activation of NodD1 and NodD2 by different inducing compounds produced at defined stages of symbiotic infection. We identified Lotus chalcone isomerase CHI4 as a root hair induced candidate involved in the biosynthesis of an inducer compound that may be preferentially recognised by NodD1 within root hair infection threads. We propose an alternative explanation for the function of multiple copies of nodD that provides the host plant with another level of compatibility scrutiny at the stage of infection thread development. This article is protected by copyright. All rights reserved.