The grim find comes as a summit on the growing European migrant crisis takes place in the Austrian capital, Vienna.
Austrian police initially thought a truck stopped on the side of the motorway had been in an accident.
But when they looked closer, they noticed blood seeping out of the back of the truck.
Inside, they found dozens of bodies.
Police Chief Hans Peter Doskozil says they are still hunting for the driver.
"We can assume that possibly 20 people have been killed, but it could also be 40 or 50. Because they've been dead for some time, and the decomposition process has already begun, at this point we can't make any concrete assertions about how they died."
Police say those inside the truck may have been dead for up to two days - before the truck had even crossed into Austria.
Austrian Interior Minister Johanna Milk-Leitner says it shows, once again, that human traffickers have no respect for the lives of their victims.
"Today is a dark day, and our thoughts are with the victims, the families of the victims and their friends. This tragedy touches us all. We all know that the people traffickers are criminals, and whoever thinks that they are there to help victims, are beyond help."
However, advocates, including this woman, are calling for political leaders to do more to ensure safe passage for migrants.
"Building up our borders or fighting ever more against people-smuggling cannot be be the solution. We need safe passages for these people. These people have nothing to lose, their choice is between taking the chance and coming to Europe or dying at home."
Leaders have met in Vienna to discuss the growing migrant crisis.
Austria says the EU has failed to address the problem of people entering via the Western Balkans.
German Chancellor Angela Merkel says the crisis can only be solved if all EU countries share responsibility.
A record number of 107,500 migrants crossed the EU's borders last month, and Serbian police counted more than 3,000 crossing into Serbia in a single day this week.
United Nations refugee agency spokeswoman Melissa Flemming says all countries need to play their part.
"What we have is a need to start in the countries where they're coming, and to receive them properly, register them properly, and to implement the relocation plan that the European Union has before it, in order to stop these awful chaotic scenes at borders."
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