Due to the clinical importance of differentiating the two species of the Entamoeba histolytica/Entamoeba dispar complex, we developed a multiplex polymerase chain reaction (PCR) method that overcomes time-consuming and laborious procedures. We report here a DNA extraction protocol using non-fixed stool samples that avoid long lysis-incubation periods through the combined use of zirconium beads and a lysis-supporting buffer. We characterized 49 of 52 stool specimens from Cuban patients with amoebiosis. Among them, 36 (75.5%) were infected only with E. dispar (the nonpathogenic species), while 13 (24.5%) displayed a mixed infection with both E. dispar and E. histolytica. The multiplex PCR protocol showed a specificity of 1.00 and a sensitivity of 0.94. Furthermore, the entire procedure can be performed in one day. This approach is therefore reliable and applicable in the field for epidemiologic studies.