The root lesion nematode (RLN) Pratylenchus penetrans is a migratory species of plant parasitic nematodes that attacks a wide range of crops including alfalfa, the third most valuable field crop in the U.S. High levels of infection can reduce alfalfa forage yields and the plant’s ability to take up water and nutrients. Currently, there are no commercially certified varieties with resistance to RLN. To advance our understanding of the host-pathogen interactions and to gain biological insights into the genetics and genomics of host resistance to RLN, we performed a comprehensive assessment of resistant and susceptible interactions of alfalfa with P. penetrans that included root penetration studies, ultrastructural observations and global gene expression profiling of host plants and the nematode. Several gene-candidates associated with alfalfa resistance to P. penetrans and nematode parasitism genes encoding nematode effector proteins were identified for potential use in alfalfa breeding programs or development of new nematicides, respectively. The induction of host genes involved in biosynthesis and accumulation of secondary metabolites in the cells, especially of the phenylpropanoid pathway, appears to be critical for alfalfa resistance to P. penetrans. We hypothesize that constitutive defenses, such as significant accumulation of tannin-like deposits in root cells of the resistant cultivar, could be a key mechanism of alfalfa resistance against P. penetrans.