As said, 500 years is just a blink in the eye for evolution; at least for a creature with life cycles as long as humans. It's generally too small to have any evolutionarily interesting changes.
However, to meet your answer, yes it is theoretically possible, though quite improbable. A species is defined as two creatures that can not reproduce with each other to create fertile offspring. If the two were incapable of reproducing they are a different species, even if they look identical in every other way. In theory as little as one mutation is all that is required to become a separate species.
In fact, that's the only reason humans are not the same species as the bonobo and the chimpanzee; two of our chromosomes merged together into a single larger one, causing a different number of chromosomes than the chimp or the bonobo had. If it wasn't for that one change it's possible we would still be able to interbreed with our closest genetic relatives.
Of course, the problem is having the entire population of Mars gain this gene, which is sort of impossible. Since they can only have children so fast, their genetics can only spread so quickly. One person with a mutation when they found Mars can not spread that mutation to a large percentage of the population in 500 years, unless, he, or one of his immediate descendents, started some truly extensive harem/breeding program to father all the next generation's population, or the population numbers were so absurdly low that inbreeding would almost certainly lead to death of the population.
This leads to improbable ways to technically create speciation. Both would only work if something made this mutation quite important, if not mandatory. In other words, your Martians would need to be facing extinction, or something close to it, that this one mutation can somehow fix. The most likely form of extinction would be that the Martians can't have children for some reason. Breeding issues, and this mutation, allowed them to get around whatever was preventing them from having children, by modifying the way they reproduced.
Intentional genetic mutation, the most likely. Facing an extinction level event on Mars, the Martians created a way to use a retrovirus, or controlled IVF, to get around the extinction level event, providing their children with a chance to survive. A side effect of this life saving treatment was to tweak their genetics in such a way to be incompatible with earth genetics; likely creating offspring that are infertile (that's still specization by definition).
Near-extinction. The colonists did not intentionally do anything to their genetics, or lacked a means to do so, and thus almost all of them died out. The ones that did survive managed to do so based off a random mutation that just happened to appear at the same time (note, I can't say how absurdly unlikely this is). This one person with the mutation spreads it to the much smaller martian population. Perhaps he is the only person capable of reliably fathering children due to some environmental radiation whatever and so by necessity is the father of all future children. Though I can't say how improbable this is; and ultimately a society like this would still be lowly dying to inbreeding.
In either case the two species would look and act almost identical. Their ability to reproduce being the only real distinction between the two. However, that is all it takes to technically be a new species.
Given more time this specialization could occur, but by more time we're talking tens of thousands of years at least, if science doesn't get involved to accelerate the process.