In the current work, samples of Mytilus galloprovincialis collected in different sites from Ría de Arousa estuary (Galicia, north-western Spain) were analysed for total Al, As, Ba, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn, and for Ba, Cu, Mn and Zn bound to metallothionein-like proteins isoform I (MLP-I) contents. Inductively coupled plasma-optical emission spectrometry (ICP-OES) was used to assess total metal contents in raft mussels, while high performance liquid chromatography (HPLC) with an on-line metal detection with ICP-OES was used to measure metals bound to MLP-I. A microwave assisted acid digestion procedure was used as a sample pre-treatment for total metal contents, while a cytosolic preparation method based on a blending procedure with TRIS–HCl (pH 7.4) as an extracting solution was used to isolate MLP-I. Concentrations of total metals and metals bound to MLP-I were used as discriminating factors to establish different regions in the Ría de Arousa estuary. Principal component analysis (PCA) and cluster analysis (CA) were used as unsupervised pattern recognition procedures, and the half-range central value transformation was used as a data pre-treatment to homogenize data sets. Results have revealed a separation of raft mussels in good agreement with water circulation pattern and oceanographic processes in the estuary only when concentrations of metals to bound MLP-I are used as discriminating factors. Otherwise, raft mussels are classified as samples harvested in the inner or outer sides of the Ría.