We have previously demonstrated that mutated DNA derived from the circulation can be detected in urine and predominantly exists as DNA fragments <1 kb. To preferentially isolate the trans‐renal DNA from urine, we developed a method using carboxylated beads to separate high‐MW (1 kb or larger) from low‐MW DNA in urine. A primer set for 18s rRNA (generating a PCR product of 872 bp) was designed and optimized for real‐time PCR quantification of high‐MW DNA templates. To evaluate the method, urine samples from 5 volunteers with no known diseases and 36 patients with various colorectal diseases were collected and tested. It was found that the average removal efficiency of high‐MW DNA from total urine DNA using carboxylated beads is 92.72%± 1.42%. Furthermore, compared with using total urine DNA, our method provides a greater ability to detect mutated K‐ras in the urine of colorectal cancer patients. The concurrence of K‐ras mutations detected in disease tissue and the corresponding urine specimen is significantly higher (P= 0.0015) when the samples were enriched in low‐MW DNA.