Advances in CT hardware have propelled the development of novel CT contrast agents. Combined with the spectral capabilities of X-ray CT, molecular imaging is possible using multiple heavy-metal contrast agents. Nanoparticle platforms make particularly attractive agents because of (1) their ability to carry a large payload of imaging moieties, and (2) their ease of surface modification to facilitate molecular targeting. While several novel imaging moieties based on high atomic number elements are being explored, iodine (I) and gadolinium (Gd) are particularly attractive because they are already in clinical use. In this work, we investigate the feasibility for in vivo discrimination of iodine and gadolinium nanoparticles using dual energy micro-CT. Phantom experiments were performed to measure the CT enhancement for I and Gd over a range of voltages from 40 to 80 kVp using a dual-source micro-CT system with energy integrating detectors having cesium iodide scintillators. The two voltages that provide maximum discrimination between I and Gd were determined to be 50 kVp with Cu filtration and 40 kVp without any filtration. Serial dilutions of I and Gd agents were imaged to determine detection sensitivity using the optimal acquisition parameters. Next, an in vivo longitudinal small animal study was performed using Liposomal I (Lip-I) and Liposomal Gd (Lip-Gd) nanoparticles. The mouse was intravenously administered Lip-Gd and imaged within 1 h post-contrast to visualize Gd in the vascular compartment. The animal was reimaged at 72 h post-contrast with dual-energy micro-CT at 40 kVp and 50 kVp to visualize the accumulation of Lip-Gd in the liver and spleen. Immediately thereafter, the animal was intravenously administered Lip-I and re-imaged. The dual energy sets were used to estimate the concentrations of Gd and I via a two-material decomposition with a non-negativity constraint. The phantom results indicated that the relative contrast enhancement per mg/ml of I to Gd was 0.85 at 40 kVp and 1.79 at 50 kVp. According to the Rose criterion (CNR<5), the detectability limits were 2.67 mg/ml for I and 2.46 mg/ml for Gd. The concentration maps confirmed the expected biodistribution, with Gd concentrated in the spleen and with I in the vasculature of the kidney, liver, and spleen. Iterative reconstruction provided higher sensitivity to detect relatively low concentrations of gadolinium. In conclusion, dual energy micro-CT can be used to discriminate and simultaneously image probes containing I and Gd.