The genetic sexing of animals having long gestation periods offers significant benefits in regard to breeding management among their populations living in captivity. In our study, a new increased-sensitivity PCR method for fetal sexing was developed and tested successfully on elephants, from only a small volume of maternal plasma. Suitable sensitivity was obtained by using short, reduced amplicon lengths with fluorescent labelling for capillary electrophoresis detection. The fundamental principle for this technique was based on the detection of two Y-specific markers (AmelY and SRY), the presence of which indicates the mother is carrying a male fetus and the absence of these markers designates a female fetus. As a reaction control, the X-chromosomal marker (PlpX) was used. To the best of our knowledge, this is the first report on this topic, confirming the presence of fetal cell-free DNA from the plasma of a pregnant captive elephant, and demonstrating a new opportunity for non-invasive assessment in fetal sex determination.