For the recent years, the application of treosulfan (TREO)-based conditioning prior to hematopoietic stem cell transplantation (HSCT) has been increasing as an alternative to busulfan-based therapy, especially for patients presenting high risk of developing hepato-, pulmo-, and neurotoxicity. So far, the penetration of TREO and its epoxy-derivatives into central nervous system and aqueous humor of the eye has been investigated. However, lacking knowledge on the compounds distribution into the other key tissues precludes comprehensive understanding and assessment of TREO clinical efficacy and toxicity. In this paper, the disposition of TREO and its active monoepoxide (S,S-EBDM) in a bone marrow, liver, lungs, brain, and quadriceps femoris was studied in an animal model. Male and female adult Wistar rats (n = 48/48) received an intraperitoneal injection of TREO at the dose of 500 mg/kg b.w. Concentrations of TREO and S,S-EBDM in tissues were determined with a validated HPLC-MS/MS method. Pharmacokinetic calculations were performed in WinNonlin using a noncompartmental analysis. Mean values of the maximal concentrations of TREO and S,S-EBDM in the organs were sex-independent and ranged from 61 to 1650 μM and 25–105 μM, respectively. No quantifiable levels of S,S-EBDM were found in the liver. Average tissue/plasma area under the curve (AUC) ratio for unbound TREO increased in the sequence: brain (0.10) < muscle (0.77) < bone marrow = lungs (0.82) < liver (0.96). The tissue/plasma AUC ratio for unbound S,S-EBDM changed as follows: brain (0.35) < lungs (0.50) < bone marrow (0.75) < muscle (1.14). Elimination half-lives of the compounds in plasma and the organs ranged from 0.7 h to 2.1 h. Scaling of the obtained AUCs of TREO and S,S-EBDM and the literature AUCs of busulfan to concentrations of the drugs in HSCT patients' plasma show that TREO reaches much higher levels in the organs than busulfan. Nonetheless, low S,S-EBDM exposure in a liver, lungs, and brain, even compared with busulfan, may contribute to relatively low organ toxicity of TREO-based conditioning regimens. Similarity of the scaled bone marrow AUCs of S,S-EBDM and busulfan corresponds to comparable myeloablative potency of TREO- and busulfan-based conditioning. The biological half-lives of TREO and S,S-EBDM in plasma and the studied organs indicate that 48 h lag time following administration of the last dose of TREO to HSCT patients is sufficient to protect the transplanted stem cells from the compounds' exposure.