Phyllanthus niruri is known as a stone breaker and has some biological activities such as antioxidant, antibacterial, anti-inflammatory, antiviral, immunostimulant, etc. There are two other species related to P. niruri namely P. debilis and P. urinaria. The morphology of the three plants is quite similar so that identification and discrimination of the three species are very crucial to avoid adulteration of P. niruri from the two related species for ensuring the quality, efficacy, authenticity, and safety of its herbal medicinal product. This study aims to develop identification and discrimination method of P. niruri based on thin-layer chromatographic (TLC) fingerprint combined with chemometrics analysis. The result showed that P. niruriprovides a different fingerprint profile compare to P. debilis and P. urinaria. P. niruri has two characteristic bands (Rf 0.71 and 0.77) that could be used for discrimination from P. debilis and P. urinaria. To enhance the discrimination of the three species, we used discriminant analysis with peak area from 13 detected peaks as the variable. From the results obtained, the discriminant analysis showed the separation of each species using the first two discriminant function (DF) with variance for DF-1 = 63.34% and DF-2 = 36.66% in DA plots, which explain 100% of total discriminant factor varianceand correct classification rate is 100%. The developed TLC fingerprint combined with discriminant analysis successfully distinguished three closely related species of Phyllanthus.