Purpose: Increased radiation dose for recently developed clinical total marrow irradiation (TMI) facilitates bone marrow transplants for high risk leukemia patients but its mechanism and role is completely unknown. To initiate this high impact, clinically relevant, investigation, a murine TMI‐like system has been developed and benchmarked. Detailed dosimetric characterization of the irradiator, toxicity to critical organs, and molecular imaging biomarkers were evaluated. Methods: An orthovoltage x‐ray unit was commissioned and calibrated following TG‐61 protocol. A special irradiation jig utilizing compensators has been developed to reduce the dose to eyes, brain, lungs, heart, and gastrointestinal tract. Validation of the dosimetry has been performed using TLDs, Gafchromic film, ionization chamber, and Monte Carlo calculations. Mice were irradiated for TBI and TMI. Live and longitudinal pre and post radiation (day 2) FDG microPET/CT imaging was performed. Certain organs such as lungs, liver, kidney, gut and brain were harvested post radiation for the purpose of cellular and histological studies using thymidine analog 5‐bromo‐2‐deoxyuridine (BrdU). Results: The dose is verified by performing TLD and film dosimetry on the entrance and exit sides, as well as placing dosimeters inside the subjects. Both absolute doses and planar dose distributions have been determined providing an extensive set of data representing the dose within the subjects. Dose to critical organs was reduced by a factor of two in TMI cases. As a Result metabolic and cellular activities were reduced in TMI mice at the GI and brain compared to TBI mice. Bone marrow proliferation remains the same as bone marrow dose is unchanged. Conclusion: Properly characterized x‐ray beam, accurate dosimetry, and detailed molecular and cellular studies are of importance in radiobiology investigations in support of TMI model which may lead to new therapies for humans. Reduction in side effects such as GI toxicity from radiation is promising for transplantation. This project is supported by NIH.