Disruptor of Telomeric Silencing 1-Like (DOT1L), the sole histone H3 lysine 79 (H3K79) methyltransferase, is required for leukemogenic transformation in a subset of leukemias bearing chromosomal translocations of the Mixed Lineage Leukemia (MLL) gene, as well as other cancers. Thus, DOT1L is an attractive therapeutic target and discovery of small molecule inhibitors remain of high interest. Herein, we are presenting screening results for a unique focused library of 1200 nucleoside analogs originally produced under the aegis of the NIH Pilot Scale Library Program. The complete nucleoside set was screened virtually against DOT1L, resulting in 210 putative hits. In vitro screening of the virtual hits resulted in validation of 11 compounds as DOT1L inhibitors clustered into two distinct chemical classes, adenosine-based inhibitors and a new chemotype that lacks adenosine. Based on the developed DOT1L ligand binding model, a structure-based design strategy was applied and a second-generation of non-nucleoside DOT1L inhibitors was developed. Newly synthesized compound 25 was the most potent DOT1L inhibitor in the new series with an IC50 of 1.0 μM, showing 40-fold improvement in comparison with hit 9 and exhibiting reasonable on target effects in a DOT1L dependent murine cell line. These compounds represent novel chemical probes with a unique non-nucleoside scaffold that bind and compete with the SAM binding site of DOT1L, thus providing foundation for further medicinal chemistry efforts to develop more potent compounds.