Sulfur (₁₆S) has 23 known isotopes with mass numbers ranging from 27 to 49, four of which are stable: ³²S (95.02%), ³³S (0.75%), ³⁴S (4.21%), and ³⁶S (0.02%). The preponderance of sulfur-32 is explained by its production from carbon-12 plus successive fusion capture of five helium-4 nuclei, in the so-called alpha process of exploding type II supernovas (see silicon burning).

Other than ³⁵S, the radioactive isotopes of sulfur are all comparatively short-lived. ³⁵S is formed from cosmic ray spallation of ⁴⁰Ar in the atmosphere. It has a half-life of 87 days. The next longest-lived radioisotope is sulfur-38, with a half-life of 170 minutes. The shortest-lived is ⁴⁹S, with a half-life shorter than 200 nanoseconds. Heavier radioactive isotopes of sulfur decay to chlorine.

When sulfide minerals are precipitated, isotopic equilibration among solids and liquid may cause small differences in the δ³⁴S values of co-genetic minerals. The differences between minerals can be used to estimate the temperature of equilibration. The δ¹³C and δ³⁴S of coexisting carbonates and sulfides can be used to determine the pH and oxygen fugacity of the ore-bearing fluid during ore formation.

In most forest ecosystems, sulfate is derived mostly from the atmosphere; weathering of ore minerals and evaporites also contribute some sulfur. Sulfur with a distinctive isotopic composition has been used to identify pollution sources, and enriched sulfur has been added as a tracer in hydrologic studies. Differences in the natural abundances can also be used in systems where there is sufficient variation in the ³⁴S of ecosystem components. Rocky Mountain lakes thought to be dominated by atmospheric sources of sulfate have been found to have different δ³⁴S values from oceans believed to be dominated by watershed sources of sulfate.