Nuclear DNA is widely used to estimate phylogenetic and phylogeographic relationships. Nuclear gene variants may be present in an individual's genome, and these result in Intra-Individual Site Polymorphisms (2ISP; pronounced 'twisp') in direct-PCR or individual-consensus sequences based on a clone sample. 2ISPs can occur fairly often, especially within, but not restricted to, high-copy-number regions such as the widely used internal transcribed spacers of the nuclear ribosomal cistron. Dealing with 2ISPs has been problematic as phylogeny reconstruction optimality criteria generally do not take account of this variation. Here we test whether an approach that treats 2ISPs as additional (termed 'informative'), rather than ambiguous, characters offers improved support in three common criteria used for phylogenetic inference: Minimum Evolution (via Neighbour Joining), Maximum Parsimony and Maximum Likelihood. We demonstrate significant improvements using the 2ISP-informative treatment with simulated, real-world and case study datasets. We envisage that this 2ISP-informative approach will greatly aid phylogenetic inference using any nuclear DNA regions that contain polymorphisms within individuals (including consensus sequences generated from next generation sequencing), especially at the intrageneric or intraspecific level.