OBJECTIVES: We estimated the incremental cost-utility of newer chromogenic or silver in situ hybridization (CISH, SISH) techniques vs. fluorescence-based methods (FISH) to diagnose human epidermal growth factor receptor-2 positive (HER2+) breast cancer (BC) and direct targeted adjuvant trastuzumab using decision-analytic modelling. Newer CISH and SISH may be more widely useful in pathology practice owing to the use of light (vs. fluorescence) microscopy.   METHODS: A decision tree represented 6 alternative test-treat strategies to direct adjuvant trastuzumab in HER2+ early-stage BC. The strategies consisted of primary CISH, FISH or SISH testing, or, primary immunohistochemistry (IHC) with CISH, FISH or SISH confirmation of IHC equivocal results only. We assumed FISH as gold standard, with trastuzumab treatment for IHC+ or FISH+. A Markov model of early-stage BC estimated the sequela of treatment based on test results by accounting for true HER2 status in the lifetime horizon from the payer’s perspective.  Treatment effect and test accuracy parameters were informed by meta-analyses. Uncertainty was characterized using probabilistic sensitivity analyses and cost-effectiveness acceptability curves.   RESULTS: Primary FISH testing was dominant in the base case, assuming 20% HER2+ disease prevalence, decreasing costs by a mean of $815 and improving outcomes by 0.0022 QALYS compared to the referent strategy of primary IHC with FISH confirmation of IHC equivocal results. After HER2+ disease prevalence, results were most sensitive to test sensitivity and specificity parameters.  Primary FISH, CISH or SISH strategies had higher probabilities of being cost-effective than primary IHC strategies.   CONCLUSIONS: Our results suggest that the additional costs of primary in situ hybridization testing are offset in the long-term by improved health outcomes.  The significant uncertainty attributed to test accuracy parameters suggests that additional research is needed to determine whether newer CISH or SISH can perform equivalently to FISH for HER2 status determination.