A quick polymerase chain reaction (PCR) assay was developed for the detection of Leifsonia xyli subsp. xyli (Lxx), the bacterial causal agent of ratoon stunting disease (RSD) of sugarcane, in xylem sap samples from stalks. After removal of abiotic impurities and large molecular weight microorganisms by a 3000 rpm, 5 min centrifugation, the Lxx bacteria were precipitated from the xylem sap by a 12,000 rpm, 10 min centrifugation. The Lxx cells were re-suspended in 50 μl freshly prepared Buffer A (0.1M NaOH + 2% Tween 20), lysed by heating at 95°C for 10 min, cooled down on ice for 3 min, and neutralized by mixing with 50 μl of freshly prepared Buffer B (0.1M Tris-HCl, pH 8.0 + 2 mM EDTA). The resulting lysates were directly subjected to conventional PCR with Lxx-specific primers. Of the 31 varieties tested, 19 were positive for Lxx infection, including all FN, GT, and YT varieties. These varieties also tested positive by PCR on DNA templates extracted from xylem sap samples using a CTAB procedure and by two enzyme-immunoassays, dot-blot (DBEIA) and direct antigen coating (DAC-ELISA). DB-EIA and DAC-ELISA detected Lxx in 90.3 and 93.5%, respectively, of the varieties while the detection rate with PCR was 61.3%. The modified PCR assay was quick and more economical. It did not require organic solvent usage or ethanol precipitation, but produced the same level of detection as that of the PCR using DNA samples prepared by the CTAB procedure. All the PCR amplicons were 439 bp in size sharing the same nucleotide sequence. This consensus sequence, GenBank Accession EU723209, aligned perfectly with three other Lxx nucleotide sequences available in the GenBank, AE016822, AF034641, and DQ232616. However, two mis-matches, a (G?A) transversion and a deletion, were found in another nucleotide sequence AF056003.