Most elements exist in nature as a mixture of isotopes. The isotopes differ in weight due to their respective number of neutrons. The molecular weight of a molecule is different depending on the specific isotope of its elements involved. As a result, the mass spectrum of the molecule exhibits peaks from the same fragment at multiple positions. The positions of these mass signals depend on the difference between the molecular mass. Furthermore, the intensity of these signals is dependent on the relative ratio of isotopic abundance in nature.

For example, carbon has two stable naturally occurring isotopes of molecular mass 12 and 13 u that coexist in a relative composition of 1:0.011. The mass signals for a carbon-containing molecular ion or a charged fragment are positioned at its molecular mass (M) and one unit higher than its molecular mass (M+1). The relative ratio of the M+1 peak to M peak depends on the number of carbon atoms in the particular molecule. The M and M+1 peaks of methane, decane, and icosane are compared. As the number of carbon atoms increases, the relative intensity of the M+1 peak also increases because of the increasing likelihood of the molecule containing a ¹³C atom.

The isotope peaks in the mass spectrum and their relative intensity are used to identify the presence of specific elements in the unknown analyte. For example, an M+2 peak in the molecule indicates the presence of chlorine or bromine in the molecule, since the isotopes of halides differ by two u. Additionally, from the relative intensity of the M+2 peak, it is possible to differentiate between the chlorine and bromine in the analyte. The abundance of chlorine isotopes is 75% of ³⁵Cl and 25% of ³⁷Cl, whereas, for bromine isotopes, it is approximately 50% of ⁷⁹Br and ⁸¹Br. The difference in the relative ratio between M and M+2 peaks in the mass spectra of chlorobenzene and bromobenzene is notable.