Isotopologues are molecules that differ from the parent molecule only in their isotopic composition. For example, ordinary light water (H
2
O), semi‐heavy water (HDO), and heavy water (D
2
O) are isotopologues of the same molecule. They have the same chemical formula and bonding arrangement of atoms, but at least one atom has a different number of neutrons than the parent. Isotopologues that differ only by the location of an isotopically modified element are called isotopomers. For example, the
15
N isotope in the N
2
O molecule can be either next to the oxygen atom or second next to it; thus, N
15
NO and
15
NNO are isotopomers of the same molecule. Detecting isotopologues of different molecules is critical in such fields as astrobiology, biogeochemistry, personalized medicine, and forensics, with mass spectrometry being a major technique to distinguish between different isotopologues. In this article, I focus on a new technique for detection of isotopologues based on laser spectroscopy. The backbone of this technology is massively parallel spectroscopic probing in the mid‐infrared spectral region by a frequency comb – a broad spectrum composed of some million phase‐locked equidistant sharp spectral lines – produced by a subharmonic optical parametric oscillator (OPO). Through assessing their unique rotational‐vibrational absorption signatures, we are able to simultaneously detect numerous molecules and their isotopologues in a mixture of gases, in real time and with one part‐per‐billion detection capability.