Zimbabwean Hatiwande Nyamande took second in a time of 5:38:48, while former champion Gift Kelehe was third in 5:41:48.
Another previous champion crossed the line in the top four, with Ludwick Mamabolo placing fourth in 5:42:40.
Charles Tjiane, who led at halfway, claimed fifth place in 5:45:26.
The 2014 Comrades champion Mthembu added a second title to his growing list of accolades, which also includes a silver medal at the 100km World Championships last year.
Tjiane held the lead after 42km at Drummond Hill, with Mthembu 1:47 behind way down in 13th place.
By the 56km mark, however, Mthembu had pulled into second position, but had lost more time on the lead at 2:40 behind.
It was after that point that Mthembu made his move and by
At the infamous Polly Shortts at the 79km mark, Mthembu had opened up a healthy gap on the field, with over a minute between himself and Nyamande.
Kelehe was 2:42 behind at that stage in third.
By the time Mthembu made his way to the finish at Scottsville racecourse, he was over three minutes ahead and he was able to savour the run to the line.African News Agency (ANA)