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Formaldehyde fixation is worldwide the most used system for histopathological examination. However, its toxicity is well known, and preservation of proteins and nucleic acids is not optimal. Alternative fixatives warranting similar morphological quality of tissues and costs, but lacking toxicity and allowing better preservation of proteins and nucleic acids would therefore increase both safety of operators and quality of molecular analysis in pathology.This multi-institutional study aimed to compare the morphological, histochemical, immunohistochemical (IHC), and molecular analyses outcomes of a newly patented, non-toxic, acid-free Glyoxal (GAF) fixative with neutral buffered formaldehyde (NBF). Tissues from a total of 73 subjects were analyzed, including 13 necropsies.Gross features were preserved after GAF fixation, with no tissue hardening or discoloration. Cellular ultrastructure was also better preserved with GAF and histology and histochemistry on GAF-fixed samples showed good results when compared to NBF-fixed samples, with the exception of loss of tinctorial affinity of erythrocytes and mast cell granules. IHC analyses also showed comparable results with only slight and rare protocol adjustment. DNA and RNA yields were higher from GAF-fixed samples (Pp53andCOX1) were better amplified. RNA scope showed positive results forc-KITexpression in GAF-fixed mast cell tumors.Based on these data, the non-toxic GAF fixative allows good macroscopical, histological and immunohistochemical analyses of tissue samples, including on-field application, and better molecular analyses when compared to NBF. This represents a promising possibility for teaching, diagnostic, and research in veterinary pathology.