Pyrite has been extensively utilized for tracing geological processes through its iron and sulfur isotopemicro-analysis. However, the lack of matrix-matched reference materials with characterized Fe and Sisotopic compositions poses a challenge. In this study, we examined a natural pyrite sample (IGGPy-1)to evaluate its potential as a matrix-matched reference material for in situ micro-analysis of iron andsulfur isotopes. Electron probe microanalysis (EPMA) confirmed the homogeneity of major elementswithout any growth zoning in pyrite grains. Random spot isotopic analyses conducted using laserablation multi-collector inductively coupled plasma mass spectrometry (LA-MC-ICP-MS) yieldedvariances in d56Fe and d34S of 0.07& (2S, N = 105) and 0.13& (2S, N = 105), respectively. Based on theresults and statistical analysis, it was determined that the IGGPy-1 sample was isotopicallyhomogeneous and had the potential to be used as a reference material. The d34SVCDT value, measuredusing elemental analyzer-isotope ratio mass spectrometry (EA-IRMS), was 17.09 ± 0.30& (2S, N = 6).Additionally, d56FeIRMM-014 and d57FeIRMM-014 values, measured using solution-nebulizing multicollector inductively coupled plasma mass spectrometry (SN-MC-ICP-MS), were −1.31 ± 0.06& (2S, N= 21) and −1.94 ± 0.12& (2S, N = 21), respectively. These values are recommended as the isotopicreference values for IGGPy-1. Since the samples with complex core-rim textures resulting frommultiple stages in the formation of many sulfide deposits face a significant risk of mismatch in the Feand S isotopic data obtained from different analytical domains, we presented the technique ofsimultaneous in situ measurements of Fe–S isotopes in pyrite using a single laser spot by couplinga femtosecond laser ablation system with two sets of MC-ICP-MS. Furthermore, our results clearlydemonstrated the significant impact of position effects on the accuracy of Fe and S isotopic data.Therefore, it is crucial to identify appropriate domains within the ablation chamber for placing the“standard” and samples for mitigating these effects.