Methods for fast, simple detection of biomarkers to detect and differentiate closely related Bacillus endospores including Bacillus anthracis (BA), Bacillus thuringiensis (BT), Bacillus atrophaeus (BG), and Bacillus cereus (BC) using thermochemolysis and methylation (TCM), coiled wire filament (CWF), solid phase micro extraction (SPME) and gas chromatography-mass spectrometry (GC-MS) were developed. The main biomarkers detected and used for differentiation include dipicolinic acid methyl ester (DPAME), fatty acid methyl esters (FAMEs), 3-methyl-2-butenoic acid methyl ester (3M2BAME), 2-butenoic acid methyl ester (2BAME), and several methylated sugars. TCM of endospores was performed based on hydrolysis and methylation at elevated temperature after the endospores were mixed with sulfuric acid (H2SO4) with or without addition of tetramethylammonium hydroxide (TMAH) in methanol (MeOH). TCM products were then introduced into a heated GC injector port using a coiled wire filament (CWF) or solid phase microextraction (SPME) for detection and differentiation of the endospores by GC-MS.The CWF, which consisted of a tiny platinum helical wire coil attached to a retractable plunger that moved the coil in and out of a syringe needle housing, allowed for sampling to be accomplished by dipping the CWF in an endospore sample suspension, evaporating the suspension liquid, and then introducing the CWF into the injection port to enable on-line TCM. New SPME techniques, including half-half extraction, coated-needle extraction (CNE), and a new home-made polymer coated needle were used to speed up solid phase micro extraction of biomarkers produced from TCM. These simplified the detection of anthrose and other biomarkers. TCM with a CWF and TCM with SPME produced high intensity profiles of DPAME, FAMEs, 2BAME, 3M2BAME and methylated sugars. While the presence of DPAME can be used for the general detection of endospores (Bacillus and Clostridium) and the presence of 3M2BAME for the detection of BA, specific saturated and unsaturated C15, C16, and C17 fatty acid methyl esters and methylated sugars provide additional information for differentiating various Bacillus species grown at different temperatures and in different media. DPAME was detected in samples containing as few as 2,500 and 6,000 endospores using TCM-CWF with and without a concentration step, respectively. GC-MS peak area percent reproducibility for FAMEs using TCM and CWF varied from 3 to 13% (RSD). Better than 97% correct predictability of Bacillus species identity was obtained from a blind experiment consisting of 145 samples using DPAME and specific FAMEs. Conventional SPME and a modified form of "in-needle" extraction allow for detection of the biomarkers in less than 35 min. The detection limits with SPME sample introduction injection were approximately 5 x 103 endospores.Using these approaches, differentiation of Bacillus endospores and other biological agents grown under different conditions were based on the following characteristics: (1) presence of DPAME and specific FAMEs (iso or anteiso C15:0 and iso or anteiso C17:0) in Bacillus endospores, (2) unique presence of 3M2BAME (anthrose by-product) in BA, (3) absence of 2-butenoic acid methyl ester in BG, and (4) presence and absence of specific methylated monosaccharides in various Bacillus species. Clostridium endospores and non-sporulating bacteria, such as Yersinia pestis (YP) and Francisella tularensis (FT) could also be easily distinguished from Bacillus endospores based on the presence and absence of several specific sugar derivatives and fatty acid methyl esters (FAMEs), such as iso or anteiso C15:0 and iso or anteiso C17:0, and > C18 FAMEs which were simultaneously produced during TCM.