The development of a simple and cost-effective method to map the distribution of RNA polymerase II (RNPII) genome-wide at a high resolution is highly beneficial to study cellular transcriptional activity. Here we report a mutation-based and enrichment-free global chromatin run-on sequencing (mGRO-seq) technique to locate active RNPII sites genome-wide at near-base resolution. An adenosine triphosphate (ATP) analog named N6-allyladenosine triphosphate (a6ATP) was designed and could be incorporated into nascent RNAs at RNPII-located positions during a chromatin run-on reaction. By treatment of the run-on RNAs with a mild iodination reaction and subjection of the products to reverse transcription into complementary DNA (cDNA), base mismatch occurs at the original a6A incorporation sites, thus making the RNPII locations detected in the high-throughput cDNA sequencing. The mGRO-seq yields both the map of RNPII sites and the chromatin RNA abundance and holds great promise for the study of single-cell transcriptional activity.