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Abstract Background: Fragments of DNA from cells dying throughout the body are detectable in urine (transrenal DNA, or Tr-DNA). Our goal was the optimization of Tr-DNA isolation and detection techniques, using as a model the analysis of fetal DNA in maternal urine. Methods: We isolated urinary DNA using a traditional silica-based method and using a new technique based on adsorption of cell-free nucleic acids on Q-Sepharose resin. The presence of Y chromosome–specific SRY (sex-determining region Y) sequences in urine of pregnant women was detected by conventional and real-time PCR using primers/probe sets designed for 25-, 39-, 65-, and 88-bp PCR targets. Results: Method of DNA isolation and PCR target size affected fetal Tr-DNA detection. Assay diagnostic sensitivity increases as the PCR target is shortened. Shorter DNA fragments (50–150 bp) could be isolated by Q-resin–based technique, which also facilitated fetal Tr-DNA analysis. Using DNA isolated by Q-resin–based method and an “ultrashort” DNA target, we successfully detected SRY sequences in 78 of 82 urine samples from women pregnant with male fetuses (positive predictive value 87.6%). Eleven of 91 urine samples from women pregnant with female fetuses produced SRY false-positive results (negative predictive value 95.2%). Conclusions: Single-copy fetal DNA sequences can be successfully detected in the urine of pregnant women when adequate methods for DNA isolation and analysis are applied. Strong precautions against sample contamination with male cells and DNA are necessary to avoid false-positive results.