The spots appear on the side of Pluto that always faces its largest moon Charon - the face that will be invisible to New Horizons when the spacecraft makes its close flyby on July 14.
The large dark areas are estimated to be 300 miles across, an area roughly the size of the state of Missouri.
The image is the last, best look that anyone will have of Pluto's far side for decades to come, said Alan Stern, New Horizons principal investigator from Southwest Research Institute in Colorado.
The spots are connected to a dark belt that circles Pluto's equatorial region.
It is weird that they are spaced so regularly, added New Horizons programme scientist Curt Niebur.
"We cannot tell whether they are plateaus or plains or whether they are brightness variations on a completely smooth surface," said Jeff Moore of Nasa's Ames Research Center in California.
In comparison with earlier images, the dark areas are more complex than they initially appeared, while the boundaries between the dark and bright terrains are irregular and sharply defined.
New Horizons is currently three billion miles away from the Earth and just two-and-half million miles from Pluto.
On July 14, New Horizons will pass about 12,500 km from Pluto.
It will zip past Pluto at 49,600 km per hour, with a suite of seven science instruments busily gathering data.
The mission will complete the initial reconnaissance of the solar system with the first-ever look at the icy dwarf planet.
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