From detailed study of 1D and 2D NMR spectra of ten natural 1,2-epoxyguaianolides (bis-1,2:3,4-epoxyguaianolides and guaianolide-1,2-epoxychlorohydrins), we identified general spectral traits helpful for stereochemical assignment of such sesquiterpene lactones. We found that the chemical shifts of certain (1)H and (13)C nuclei are consistently dependent on the configuration of 1,2-epoxy-ring which could be used as a simple rule for establishing this configuration. Then, from 1D and 2D (COSY, NOESY, HMQC, HMBC) NMR data, applying the observed rule, the structure and stereochemistry of two new, diastereomeric guaianolide-1,2-epoxychlorohydrins, isolated from Achillea serbica, are determined. The NMR data, namely, nuclear overhauser enhancement (NOE) correlations, pointed out two conformations of guaianolide's cycloheptane ring. The semiempirical calculations (AM1 and PM3 methods), performed in order to gain additional information regarding conformations, resulted in three geometries of investigated lactones. Even so, the conformations derived from the NMR data agreed well with those calculated by semiempirical methods.