Triplet formation and interactions with emissive singlet excitons are investigated in poly(3-hexylselenophene) (P3HS) using single molecule spectroscopy. P3HS is a heavy atom analog of the more commonly studied poly(3-hexylthiophene) (P3HT), a benchmark polymer for solar cells. P3HS tends to aggregate strongly which necessitates dilution to ultra-low levels within a solid inert host in order to resolve photophysical responses of single chains. Fluorescence excitation intensity modulation is performed on isolated P3HS chains using a sequence of rectangular pulses of varying intensities to probe the presence of spin-forbidden triplet excitons. Triplet population dynamics originating from singlet–triplet and triplet–triplet interactions appear as quenching of the initial fluorescence intensity to steady-state levels on characteristic time scales of ∼1–10 μs. Over 80% of all molecules studied display significant fluorescence intensity modulation (quenching depths >50%) indicative of efficient intersystem crossing and large triplet occupancies. Because triplets are highly localized and singlet–triplet and triplet–triplet annihilation rate constants are comparable to those of intersystem crossing, multiple triplets are present at any given time on single P3HS chains. Triplet lifetimes were estimated to be ∼4 μs (upper limit) determined from recovery to the ground electronic singlet state in the absence of light and, surprisingly, triplets vanish at the onset of P3HS aggregation. This result was unexpected since P3HS triplet formation takes place on time scales <30 ps making this process competitive with most accessible non-radiative deactivation pathways.