Gram-positive bacteria are those that are stained dark blue or violet by gram staining, in contrast to gram-negative bacteria, which are not affected by the stain. The stain is caused by a high amount of peptidoglycan in the cell wall, which typically, but not always lacks the secondary membrane and lipopolysaccharide layer found in Gram-negative bacteria.

In the original bacterial phyla, the gram-positive forms made up the phylum Firmicutes, a name now used for the largest group. It includes many well-known genera such as Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, and Clostridium. It has also been expanded to include the Mollicutes, bacteria like Mycoplasma that lack cell walls and so cannot be stained by Gram, but are derived from such forms.

The actinobacteria are another major group of gram-positive bacteria; they and the Firmicutes are referred to as the high and low G+C groups based on the guanosine and cytosine content of their DNA. If the second membrane is a derived condition, the two may have been basal among the bacteria, otherwise they are probably a relatively recent monophyletic group. They have been considered as possible ancestors for the archaeans and eukaryotes, both because they are unusual in lacking the second membrane and because of various biochemical similarities such as the presence of sterols.

The Deinococcus-Thermus group of bacteria also have gram-positive stains, although they are structurally similar to gram-negative bacteria.