Little is known about the load of mutations and polymorphisms in the mitochondrial DNA (mtDNA) of human oocytes and the possible effect these mutations may have during life. To investigate this, we optimised at the single cell level the recently developed method to screen the entire mtDNA for mainly heteroplasmic mutations by denaturing high performance liquid chromatography analysis. This method is sensitive (approximately 1% heteroplasmy detectable), specific and rapid. The entire mtDNA of 26 oocytes of 13 women was screened by this method. Ten different heteroplasmic mutations, of which only one was located in the D-loop and two were observed twice, were detected in seven oocytes with mutation loads ranging from <5% to 50%. From eight women >1 oocyte was received and in four of them heteroplasmic differences between oocytes of the same woman were observed. In one of these four, two homoplasmic D-loop variants were also detected. Additionally, four oocytes of a single woman were sequenced using the MitoChip (which lacks the D-loop region), but all sequences were identical. It is concluded that heteroplasmic mtDNA mutations are common in oocytes and that, depending on the position and mutation load, they might increase the risk of developing OXPHOS disease early or later in life.