Relapse to methamphetamine (Meth) seeking progressively increases after withdrawal from drug self-administration (incubation of Meth craving). We previously demonstrated a role of dorsomedial striatum (DMS) dopamine D1 receptors (D1Rs) in this incubation. Here, we studied the role of afferent glutamatergic projections into DMS and local D1R-glutamate interaction in this incubation in male rats.We first measured projection-specific activation on day 30 relapse test by using CTb (retrograde tracer) + Fos (activity marker) double-labeling in projection areas. Next, we determined the effect of pharmacological reversible inactivation of lateral or medial anterior intralaminar nuclei of thalamus (AIT-L or AIT-M) on incubated Meth seeking on withdrawal day 30. We then used an anatomical asymmetrical disconnection procedure to determine whether an interaction between AIT-L→DMS glutamatergic projections and post-synaptic DMS D1Rs contributes to incubated Meth seeking. We also determined the effect of unilateral inactivation of AIT-L and D1Rs blockade of DMS on incubated Meth seeking, and the effect of contralateral disconnection of AIT-L→DMS projections on non-incubated Meth seeking on withdrawal day 1.Incubated Meth seeking was associated with selective activation of AIT→DMS projections; other glutamatergic projections to DMS were not activated. AIT-L (but not AIT-M) inactivation or anatomical disconnection of AIT-L→DMS projections decreased incubated Meth seeking. Unilateral inactivation of AIT-L or D1Rs blockade of DMS had no effect on incubated Meth craving, and contralateral disconnection of AIT-L→DMS had no effect on non-incubated Meth seeking.Our results identify a novel role of AIT-L and AIT-L→DMS glutamatergic projections in incubation of drug craving and drug seeking.SIGNIFICANCE STATEMENTMethamphetamine seeking progressively increases after withdrawal from drug self-administration, a phenomenon termed incubation of methamphetamine craving. We previously found that D1R-mediated dopamine transmission in dorsomedial striatum plays a critical role in this incubation phenomenon. Here, we used neuroanatomical and neuropharmacological methods in rats to demonstrate that an interaction between the glutamatergic projection from the lateral anterior intralaminar nuclei of thalamus to dorsomedial striatum and local dopamine D1Rs plays a critical role in relapse to methamphetamine seeking after prolonged withdrawal. Our study identified a novel motivation-related thalamostriatal projection that is critical to relapse to drug seeking.