We present a system of image co-registration and its validation in phantom and volunteer studies. The system co-registered images via six novel non-invasive and non-radioactive external markers. The fiducial markers were attached with sponge bases on the skin surface of the phantom and the volunteers in a non-collinear and non-coplanar array. The subjects were scanned with a 1.5-T magnetic resonance (MR) imager using 2D spin-echo T1-weighted (SE) and 3D spoiled gradient recalled pulse sequences (SPGR) and with a positron emission tomography (PET) scanner for transmission imaging (TI) and emission imaging (EI). The sponge bases created radiolucent gaps with good contrast between the fiducial markers and skin surface. They made the markers visible with clear edge boundaries on both PET and MR images. The images to be registered were rescaled, interpolated, reformatted and followed by point-to-point registration for coordinate determination and the estimation of geometrical transformation and fiducial registration errors (FREs) via the fiducial markers. The images formed four matched pairs of inter-modality (SE-TI, SPGR-TI, SE-EI and SPGR-EI) and two pairs of intra-modality (SE-SPGR, TI-EI) imaging for direct co-registration. The parameters for direct co-registration of SE-TI and SPRG-TI were subsequently used as a bridge and applied for indirect co-registration of SE with EI (SE-EI(TI)) and SPGR with EI (SPGR-EI(TI)), respectively. The overall FREs of the phantom were, respectively, 1.50 mm for inter-modality and 1.10 mm for intra-modality direct co-registration. Those of volunteers were, respectively, 1.79 mm for inter-modality and 1.21 mm for intra-modality direct co-registration. For indirect co-registration, the overall FREs of the phantom were 2.53 mm (SE-EI(TI)) and 2.28 (SPGR-EI(TI)) mm; those of volunteers were 2.84 mm (SE-EI(TI)) and 2.81 mm (SPGR-EI(TI)). The errors of direct co-registration were smaller than those of indirect co-registration; the errors of phantom studies, MR-EI and SPGR-PET were smaller than those of the volunteer studies, MR-TI and SE-PET, respectively (all P<0.01, paired-difference test). In conclusion, motion artefacts, imaging blurring and spatial resolution of imaging remained the key factors affecting the accuracy of co-registration. High-accuracy indirect co-registration is provided by using non-invasive and non-radioactive external fiducial markers. The errors were less than 3 mm for both phantom and volunteer studies. The system is applicable for imaging co-registration of inter-modality non-dual imaging, inter-modality multi-tracer imaging and intra-modality multiple parameter images in clinical practice.